Eluting analytes from bead arrays

ABSTRACT

Microarray compositions suitable for analysis by one or several spectrographic methods are disclosed. In an embodiment, a microarray composition includes a three-dimensional solid support and a plurality of reactive microbeads positioned on the solid support in spatially distinct and addressable locations.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/440,332, inventor Vladislav B. Bergo, filed Feb. 23, 2017, which, inturn, is a continuation of U.S. patent application Ser. No. 14/261,024,inventor Vladislav B. Bergo, filed Apr. 24, 2014, which, in turn, claimsthe benefit of and priority to U.S. Provisional Patent Application No.61/815,772 filed on Apr. 25, 2013, the entireties of all of which arehereby incorporated herein by reference for the teachings therein.

FIELD

The embodiments disclosed herein relate generally to the field of highthroughput biological assays and more specifically to the field ofmicroarrays and analysis of microarrays by spectrographic methods. Theembodiments disclosed herein also relate to the field of live cellmicroarrays.

BACKGROUND

Biological microarrays have flexible design, high degree of multiplexingand the ability to perform measurements in a miniature format. As aresult, they have become the preferred method of analysis in biologicalresearch and various clinical applications that require screening of alarge number of samples. The microarray technology was originallydeveloped for the analysis of oligonucleotides. It has been subsequentlyextended to other biomolecules including polypeptides, proteins,antibodies, carbohydrates, lipids and small molecules. Other examples ofmicroarrays include tissue and cell arrays.

Microarrays usually feature a large number of distinct active agents,sometimes referred to as probes, which are immobilized on a flat surfaceof a 25×75×1 mm glass slide in specific locations, known as the spots.Each spot usually contains one type of a probe. Individual spots form atwo-dimensional grid or array. Linear coordinates of each spot withinsuch grid are used to determine the identity of the probe at thatposition. Consequently, the identity of a compound that interacts witheach probe, sometimes referred to as a target, may be determined basedof the specificity of the probe-target interaction. Microarrays of thistype are known as ordered arrays or printed arrays. The unambiguouscorrelation between the identity of the probe and its location on themicroarray slide is known as positional encoding.

Alternative microarray formats also exist, in which the identity of aprobe cannot be determined from its location. Such microarrays are knownas random arrays. An example of a random array is ILLUMINA® BEADARRAY™in which individual reactive microbeads are randomly placed into wellsetched on a microwell array plate. The identity of a probe in randomarrays may be determined using bead encoding and subsequent decoding,i.e. each bead carries a unique identifying label. A variety of beadencoding technologies are known in the art.

Instead of being placed on a solid support, a library of microbeads mayreact with the sample and undergo subsequent measurement by ananalytical method while suspended in a liquid medium. Such microarrayformat is known in the art as a suspension bead array or a liquid array.Flow cytometry and fluorescence—activated cell sorting (FACS) are usedfor the screening of individual beads in a suspension bead array.

The bead-based analytical platforms are commonly used to probe affinityinteractions. In a basic form of an affinity interaction assay, eachbead carries a capture agent and a bead label or a bead tag. The beadlabel is reversibly or irreversibly linked to the bead. The captureagent, or the probe, is a specific molecule or a molecular complex thathas affinity for another molecule or molecular complex, which is knownas the target. Multiple identical copies of a capture agent are attachedto each bead. The identical beads within the bead library, which carrythe same capture agent, are known as replicates. The binding of thetarget to the bead-conjugated probe is achieved by incubation of a beadlibrary with a sample suspected of containing the target, which isnormally followed by one or more wash steps in order to minimize thenon-specific binding to the beads. The target molecules bound to thebeads may be detected directly or by utilizing a secondary probe, suchas an antibody and in some cases an additional probe, such as asecondary antibody. By using bead libraries containing beads conjugatedto different capture agents, multiple targets may be probed in a singlereaction, which is known as multiplexing. Fluorescence-based analyticalmethods are widely used for detecting targets and quantifying theirrelative amounts within a microarray.

In many aspects, the bead-based multiplexed analytical technologies aresuperior to the methods, which utilize planar, i.e. two-dimensionalmicroarrays. Some advantages of the bead arrays over the conventionalplanar microarrays include the higher amount of analyte available forthe downstream detection by a specific analytical method, greaterstability of an active agent conjugated to a bead and an easilyconfigurable composition of the bead array, for example the beads may beindividually selected and combined to create a bead library suitable fora particular assay. Furthermore, the microbead screening technologiesare inherently compatible with many known methods of the solid phasesynthesis including fabrication of combinatorial libraries and synthesisof biopolymers, such as polypeptides, polysaccharides and nucleic acidsdirectly on microbeads.

In the majority of bead-based assays the analytes are measured whilestill bound to their respective microbeads. This severely limits therange of analytical methods that can be used to perform the assayreadout. In fact, most of the current readout methods utilize variousforms of optical detection, such as fluorescence and luminescence andalso radioactivity. On the other hand, mass spectrometry-basedanalytical methods, which require desorption of the analytes from thesurface, are rarely used in high-throughput bead assays and have notbeen used for analyzing bead microarrays of large magnitude. Yet, it ishighly desirable to measure analytes using hundreds of thousands ofdifferent mass channels provided by mass spectrometry in contrast toonly a few channels available with the optical detection. For example,in the proteomic applications the mass spectrometric readout may be usedto perform label-free detection, screen for post-translationalmodifications and obtain sequence information by fragmentation ofanalytes released from individual beads.

Accordingly, there is still a need for bead microarrays, which areconfigured for releasing analytes from the individual microbeads priorto the analysis step.

SUMMARY

This application describes, in one aspect, a composite microarraycomprising a three-dimensional solid support and a plurality of reactivemicrobeads or other reactive microparticles positioned on the solidsupport in spatially distinct and addressable locations. Individualmicrobeads, which may be bonded to one or several distinct activeagents, serve as the microarray reactive sites. The three-dimensionalsolid support additionally comprises a plurality of analytical sites,wherein an analytical site is fluidically coupled to a reactive site anddimensioned to accept one or more analytes, which are released from thereactive site. The analytes, which have been transferred from thereactive sites into the analytical sites, form compact and generallyuniform spots on the solid support and are analyzable by one or severalanalytical methods, such as mass spectrometry, optical spectroscopy orother methods. In an embodiment, the analytes within the disclosedmicroarrays are also analyzable while still bound to their respectivereactive sites, i.e. prior to the analyte transfer into the analyticalsites. Methods of fabrication, usage and analysis of the disclosedcomposite microarrays are also provided in this application.

In another aspect, this application describes a composite microarraycomprising a plurality of reactive microbeads positioned on a solidsupport, such as a microwell array plate. The individual microbeadsserving as the microarray reactive sites are reversibly bound to thesolid support and may be subsequently released from the solid supportfor analysis performed outside the solid support.

In a further aspect, this application describes composite microarraysconfigured for analysis of biological cells including live biologicalcells by mass spectrometry and optionally also by optical spectroscopy.In an embodiment, the described composite cell microarrays comprise aplurality of reactive sites, in which the individual reactive sites areconfigured for providing a specific quantity of biological cells for thedownstream analysis by mass spectrometry.

DESCRIPTION OF FIGURES

The presently disclosed embodiments will be further explained withreference to the attached drawings, wherein like structures are referredto by like numerals throughout the several views. The drawings shown arenot necessarily to scale, with emphasis instead generally being placedupon illustrating the principles of the presently disclosed embodiments.

FIG. 1 is a schematic representation of an embodiment microarray alsoshowing individual microbeads positioned inside individual microwells.

FIG. 2 illustrates the steps of an embodiment method of utilizing amicroarray comprising multiple microbeads positioned on a microwellplate.

FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D are a schematic representation ofa microwell within a microwell array plate, which contains a singlemicrobead.

FIG. 4A, FIG. 4B and FIG. 4C show various embodiment microwell designs,which may be useful for securing the position of a bead inside amicrowell.

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D and FIG. 5E schematically show aposition of a bead inside a microwell.

FIG. 6 is a table listing some possible encoding and reaction readoutoptions available on a microbead microarray.

FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E and FIG. 7F schematicallyshow several embodiment methods of performing a microarray reaction.

FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 8E and FIG. 8F schematicallyshow a microbead conjugated to a capture agent and a mass spectrum thatmay be acquired from such microbead.

FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E and FIG. 9F schematicallyshow a microbead conjugated to both a capture agent and a reporter agentand a mass spectrum that may be acquired from such microbead.

FIG. 10A, FIG. 10B, FIG. 10C and FIG. 10D schematically show anembodiment method of performing kinetic measurements of a chemicalreaction on a microbead microarray.

FIG. 11A, FIG. 11B and FIG. 11C schematically show a microbeadmicroarray subdivided into separate compartments using a gasketreversibly attached to the surface of the microarray.

FIG. 12 shows various possible readout options for analyzing athree-dimensional microbead microarray.

FIG. 13 schematically illustrates the possibility of performing nucleicacid analysis in using a microbead microarray.

FIG. 14A, FIG. 14B and FIG. 14C illustrate an embodiment method ofmaking an array comprising magnetic beads on a microwell array plate andsubsequently recovering a bead from the bead array.

FIG. 15A schematically shows a method of attaching a multi-well gasketto the surface of a microwell array plate.

FIG. 15B schematically shows a multi-well gasket attached to the surfaceof a microwell array plate with some wells of the multi-well gasketfilled with a liquid medium.

FIG. 15C schematically shows a process of separating a multi-well gasketfrom a microwell array plate.

FIG. 16A, FIG. 16B, FIG. 16C and FIG. 16D schematically show a smallsection of an array of biological cells on a flat-surface slide and on amicrowell array plate.

FIG. 17A, FIG. 17B and FIG. 17C schematically show a section of amicrowell array plate additionally containing multiple reactivemicrobeads and biological cells.

FIG. 18A, FIG. 18B and FIG. 18C illustrate various decoding and reactionreadout options available on a microwell-microbead cell microarray.

FIG. 19A, FIG. 19B, FIG. 19C, FIG. 19D, FIG. 19E and FIG. 19F illustratean embodiment method of binding biological cells to microbeads andanalyzing the cells by mass spectrometry

FIG. 20 schematically shows a section of a microarray composition, whichincludes a microwell plate and a multi-well gasket attached to thesurface of the microwell plate.

FIG. 21A, FIG. 21B, FIG. 21C and FIG. 21D schematically show a method ofscreening a library of bead-conjugated compounds for biological activityusing a live cell microarray on a microwell array plate.

FIG. 22 is a microphotograph of a reactive microbead positioned inside amicrowell on a microwell array plate.

FIG. 23 is a microphotograph of crystals of CHCA MALDI matrix.

FIG. 24A and FIG. 24B are microphotographs of a CHCA matrix-coatedmicrowell array plate containing multiple beads inside individualmicrowells.

FIG. 25A, FIG. 25B, FIG. 25C and FIG. 25D are MALDI TOF MS images of amicroarray measured in different mass channels.

FIG. 26A and FIG. 26B are microphotographs of microbeads initiallypositioned within a microwell and subsequently transferred outside themicrowell, onto the top surface of the microwell plate.

FIG. 27A, FIG. 27B, FIG. 27C and FIG. 27D are MALDI TOF MS images of apeptide microarray measured in different mass channels.

FIG. 28 is a series of microphotographs acquired at different focusdistance of a fluorescent analyte released from a microbead inside amicrowell and subsequently repositioned on a microwell array plate.

FIG. 29 is a series of microphotographs acquired at different focusdistance of a fluorescent analyte released from a microbead inside amicrowell and mixed with 500 nm diameter nanoparticles on a microwellarray plate.

FIG. 30 is a series of microphotographs acquired at different focusdistance of a fluorescent analyte released from a microbead inside amicrowell and subsequently mixed with CHCA MALDI matrix on a microwellarray plate. The series of microphotographs represents athree-dimensional fluorescence image of a section of a bead microarrayprior to analysis by mass spectrometry.

FIG. 31 is a microphotograph showing depletion of a layer of CHCA MALDImatrix by the ionization laser beam of a mass spectrometer.

FIG. 32 is a microphotograph showing a bead array, in which between 3and 5 fluorescently labeled beads are placed inside a single microwellon a microwell array plate.

FIG. 33 is a series of whole cell mass spectra acquired independentlyfrom the samples containing identical cells.

FIG. 34 shows two whole cell mass spectra acquired independently fromthe samples containing identical cells and the resulting differencespectrum.

FIG. 35 shows two whole cell mass spectra acquired from the samplescontaining the etoposide-treated and control cells and the resultingdifference spectrum.

FIG. 36 is a series of whole cell mass spectra acquired independentlyfrom the microbeads containing identical cells.

FIG. 37A is a microphotograph of a bead with bound cells. FIG. 37B is amicrophotograph of a section of a microwell array plate with somemicrowells occupied by a single bead. FIG. 37C shows whole cell massspectra independently acquired from different beads arrayed on amicrowell plate.

FIG. 38 is a series of microphotographs acquired at different focusdistance of a section of a live cell microarray fabricated on amicrowell array plate.

FIG. 39A is schematic layout of a live cell microarray reacted withdifferent biologically active compounds. FIG. 39B, FIG. 39C, FIG. 39Dand FIG. 39E are MALDI TOF MS images of a reacted cell microarrayacquired in different mass channels.

FIG. 40A and FIG. 40B is a series of representative mass spectraacquired from individual locations within the reacted cell microarrayschematically shown in FIG. 39A.

FIG. 41A and FIG. 41B show a microphotograph of a section of a live cellmicroarray fabricated on a microwell array plate in the absence andpresence of etoposide.

FIG. 42A is a photograph of a microwell plate made of photo-structuredglass containing a reactive bead array. FIG. 42B is a zoom-in showingpositions of individual beads within the microwells. FIG. 42C is afluorescence image of a fluorescent peptide analyte eluted from the beadarray and localized within the microwells.

FIG. 43A is a microphotograph of a bead array comprising 140-170 μmdiameter microbeads inside 250 μm diameter microwells. FIG. 43B is amicrophotograph of a bead array comprising 200-250 μm diametermicrobeads inside 250 μm diameter microwells.

FIG. 44A, FIG. 44B and FIG. 44C schematically show a method ofidentifying areas comprising a gap between a bead and a sidewall of amicrowell and subsequently acquiring mass spectrometric data from suchareas.

FIG. 45 is a MALDI TOF mass spectrum of a bead peptide mass tag.

FIG. 46A, FIG. 46B and FIG. 46C show a series of microphotographsshowing ITO-coated microwell plate fabricated from SU-8 photoresistcoated fiber optic faceplate and an image of a microbead within amicrowell acquired via optic fibers.

FIG. 47A and FIG. 47B show excitation and emission spectra offluorescent dyes used for fabrication of optically encoded bead arraysfor mass spectrometry.

FIG. 48A, FIG. 48B and FIG. 48C show a series of microphotographsacquired by three-dimensional fluorescence imaging of a section of abead array after incubating the bead array with a digestive enzyme.

FIG. 49A and FIG. 49B show fluorescence image of a section of a fiberoptic microwell array plate containing an array of MCF-7 cellsexpressing eGFP fluorescent marker.

DETAILED DESCRIPTION

The terms “array” and “microarray” may refer to a plurality of elementslocalized in spatially distinct and addressable locations on a solidsupport.

The term “reactive microarray” may refer to a plurality of active agentslocalized in spatially distinct locations on a solid support. In anembodiment, it may refer to a plurality of active agents conjugated tobeads, which may or may not be positioned on a solid support. Forexample, the beads may be placed in a liquid medium thereby forming asuspension bead array.

The term “reactive site” may refer to a spatially distinct region withina reactive microarray that contains one or several different activeagents.

The term “analytical site” may refer to a spatially distinct regionwithin a reactive microarray that is configured for accepting analytes,which have been released from a reactive site, and for analyzing thereleased analytes by one or more analytical methods.

The term “active agent” may refer to a substance or a chemicalconstituent that possesses chemical or biological activity and iscapable of reacting with a sample, which is contacted with a microarray.Non-limiting examples of the active agents are polypeptides, peptoids,peptidomimetics, proteins, carbohydrates, nucleic acids, smallmolecules, lipids, antibodies, aptamers and intermolecular complexescomprising several distinct molecules, such as protein-proteincomplexes, protein-nucleic acid complexes, protein-lipid complexes, etc.Further examples of the active agents may include intact biologicalcells including live cells, cell fragments and virus particles.

The term “analyte” may refer to a substance or a chemical constituentthat may be detected by an analytical method. For example, a molecule, amolecular fragment, a molecular complex, or singly or multiply ionizedspecies may constitute an analyte. The term “analyte” may also refer toa plurality of identical species, e.g. identical molecules that aredetected simultaneously by an analytical method.

The terms “bead” and “microbead”, which are used interchangeablythroughout this specification, may refer to a microparticle that isapproximately spherical and has a diameter greater than approximately 1micron and smaller than approximately 1 mm. It should be howeverunderstood that beads smaller than 1 micron, for example 100 nm or 500nm and beads larger than 1 mm, for example 2 mm or 5 mm may also be usedin some embodiments of the instant disclosure. Furthermore,microparticles that are not spherical, e.g. microrods or microcubes,microparticles that have irregular shape and microparticles that havecavities may be also used in some embodiments of the instant disclosure.For non-spherical microparticles, the size of the microparticle may beestimated based on its largest linear dimension. For microparticles thatexpand their volume when exposed to a particular solvent, the size ofthe microparticle may be provided for the dry form, as well as theswollen form.

The terms “microwell array plate” and “microwell plate”, which are usedinterchangeably throughout this specification, may refer to athree-dimensional solid support comprising a plurality of microwells.

The term “microwell” may refer to a topological feature such as a well,a pit, a depression and similar, in which at least one of the lineardimensions is greater than 1 micron but smaller than 1 mm. In someembodiments, wells with linear dimensions greater than 1 mm may be alsoreferred to as microwells, for example in reference to theindustry-standard 96-well or 384-well plates.

The term “encoding” generally has the same meaning as it does in thefield of bead-based analytical assays, for example as used in(Braeckmans, K., S. C. De Smedt, M. Leblans, R. Pauwels, and J.Demeester. 2002. “Encoding microcarriers: present and futuretechnologies.” Nat Rev Drug Discov 1:447-56 or Wilson, R., A. R.Cossins, and D. G. Spiller. 2006. “Encoded microcarriers forhigh-throughput multiplexed detection.” Angew Chem Int Ed Engl45:6104-17). In particular, the term “encoding” may refer to thedistinguishable features of individual beads that are distinct from theactive compounds conjugated to the beads. The term “positional encoding”generally has the same meaning as it does in the field of printedbiological microarrays; in particular it may refer to a specific featureof a microarray that enables the identity of an active agent to bedetermined based on the location of the active agent on the solidsupport.

In an embodiment, the present specification discloses a library ofmicrobeads in which individual microbeads are reactive, i.e. conjugatedto one or more active agents. The microbeads may be positioned on asolid support in spatially distinct locations thereby forming amicroarray possessing multiple reactive sites. The microbeads arrayed onthe solid support are accessible to and capable of reacting with asample contacted with the microarray. The sample may be a biologicalfluid, e.g. serum or a lysate, such as a cell lysate or a tissue lysate.Alternatively, the sample may be a digested compound or a mixture ofdigested compounds, a single purified or a partially purified compound,e.g. an enzyme, a mixture of two or more purified or partially purifiedcompounds, e.g. enzymes, or any other compound or a combination ofcompounds known to be analyzable in a microarray format or in a beadassay format. In an embodiment, the solid support is capable ofretaining individual beads in the spatially distinct locations, althoughit may not necessarily form a chemical bond with an individual bead. Inan embodiment, the solid support comprises a plurality of analyticalsites. An analytical site is a two-dimensional or a three-dimensionalregion of the solid support that is fluidically coupled to a reactivesite. The analytical site is dimensioned to accept one or more analytesreleased from the reactive site and to localize the accepted analytes onthe solid support for analysis by one or several analytical methods.

FIG. 1 is a schematic depiction of an embodiment composite microarrayaccording to the instant specification. The solid support is a microwellarray plate 101 comprising multiple microwells 102, which in anembodiment are arranged into multiple sub-arrays 103. Microbeads 104positioned inside the microwells 102 function as the microarray reactivesites. Individual microbeads may be retained inside the microwells byvarious means, as described in greater detail below. In an embodiment,the microbeads 104 are positioned below the top surface of the microwellplate 101. In an embodiment, no more than one microbead occupies asingle microwell.

FIG. 2 is a schematic depiction of an embodiment method of utilizing thecomposite microarray of the instant disclosure. Microbeads positionedinside individual microwells on a microwell plate 201 serve as themicroarray reactive sites 202. In an embodiment, an analytical site 211is a section of a microwell, which is not occupied by the bead andfluidically coupled to the microarray reactive site. In Step 1 a sample,which is contacted with the microwell plate 201, reaches individualreactive sites 202 through openings into the microwells 203 and reactswith active agents conjugated to the microbeads. The unreacted fractionof the sample may be subsequently removed, for example washed off fromthe microarray. In Step 2 one or more analytes are released from thereacted reactive sites 212 into the analytical sites 211 and becomelocalized in distinct spots 221 on the microwell array plate.

In an embodiment, the analytical site comprises an entire volume of themicrowell, which is not occupied by the bead. In an embodiment, theanalytical site is a section of the microwell that is proximal to theopening into the microwell. In an embodiment, the analytical site is asection of the microwell that is proximal to the bead. In an embodiment,the analytical site is an outer layer of the bead, a three-dimensionalregion immediately adjacent to the outer layer of the bead, or acombination of both. In an embodiment, the analytical site is a surfacesection of the microwell array plate that is proximal to the openinginto the microwell. In an embodiment, the analytical site is a sidewallof the microwell. In an embodiment, the analytical site is a bottomsurface of the microwell. In an embodiment, localization of the releasedanalytes in different regions of the microwell array plate enablesanalysis of the released analytes by different analytical methods. In anembodiment, a distance between the reactive site and the analytical siteis less than 1 mm. For example, such distance may be achieved by placinga 90 micron diameter microbead inside a 250 micron diameter microwell.In an embodiment, a distance between the reactive site and theanalytical site is less than 500 micron. In an embodiment, a distancebetween a reactive site and an analytical site is less than 100 micron.Such distance may be achieved by placing a 90 micron diameter microbeadinside a 180 micron diameter microwell. In an embodiment, a distancebetween a reactive site and an analytical site is less than 10 micron.In an embodiment, the distance between the reactive site and theanalytical site is measured as a distance between the outer edge of thereactive site and any portion of the analytical site.

Libraries of microbeads disclosed in the instant specification are alsoknown in the art as groups, sets, kits, collections, or arrays of beads.The beads are sometimes referred to as microcarriers. Individual beadswithin a bead library are bound, linked, conjugated, or otherwiseassociated with an active agent. An active agent conjugated to a bead issometimes referred to as being “immobilized” on the bead. An activeagent may be conjugated to its corresponding bead by means of one orseveral chemical bonds, molecules, or molecular complexes, which aresometimes referred to as “linkers”. For example, an antibody may beconjugated to its corresponding bead via a protein A linker, which isdirectly conjugated to the bead. The term “linker” may also encompass a“spacer”, i.e. a molecular unit positioned between the active agent andthe surface of the bead, which serves to provide optimal physicalseparation between the active agent and the bead. Alternatively, thelinker and the spacer may be disclosed as separate structures.Non-limiting examples of molecular structures suitable for use aslinkers include a sequence of Glycine residues (the so-called poly-Glylinker), an amino acid sequence comprising several Serine and Glycineresidues (Ser-Gly linker), an amino acid sequence comprising smalland/or hydrophilic amino acids, a sequence comprising single or multiplepolyethylene glycol groups (the PEG linker), an aminohexanoic acid (theAhx linker), etc. In an embodiment, a linker comprises a nucleic acidsequence, such as a DNA sequence. The association between a bead and anactive agent may be labile, i.e. the active agent or fragments of theactive agent may be released from its corresponding carrier bead underspecific conditions. For example, an active agent may be releasable fromits carrier bead if it is conjugated using a linker that isphoto-labile, acid-labile, heat-labile, or contains a chemical bond thatcan be degraded by a digestive compound. The linker, the spacer, ortheir fragments may be also releasable from the beads. In an embodiment,an active agent is positioned in a specific orientation and at aspecific distance from the bead surface to achieve optimal interactionbetween the active agent and the sample. An active agent may besometimes referred to as a reagent or a substrate, particularly inenzyme screening assays. An active agent may be also referred to as acapture agent or a capture probe, particularly in affinity bindingassays. Multiple identical molecules of an active agent are preferablypresent on individual beads. In an embodiment, at least 10 femtomole ofan active agent is present on a single bead. In an embodiment, at least10¹⁰ molecules of an active agent are present on a single bead,preferably at least 10¹¹ molecules, more preferably at least 10¹²molecules. In an embodiment, the amount of an active agent releasablefrom a single bead is sufficient for analysis by mass spectrometry. Theanalysis by mass spectrometry may comprise detection of a molecular ion,detection of a multiply charged ion, detection of an adduct ion,detection of an ion generated by molecular fragmentation of the activeagent, as well as other types of detection. Either one type of activeagent or several distinct active agents may be conjugated to a singlebead.

The microbeads suitable for fabrication of the microarrays disclosed inthe present specification may be manufactured from a variety ofmaterials, e.g. agarose including various types of cross-linked agarose,polymers such as polyethylene, polystyrene, polymethylmethacrylate, andpolyacrylamide, cellulose, silica, silicon, glass, metals, hydrogels.The beads may comprise composite materials such as PEG-coatedpolystyrene beads or polymer-coated silica beads. The beads may havemagnetic properties, e.g. have ferromagnetic or paramagnetic properties,which will allow bead manipulation using a magnet. The beads may beporous, e.g. possess nanopores for analyte fractionation based on thesize exclusion technology. Nanoporous controlled pore glass beads havebeen previously used for selective enrichment of a subset of low MWproteins and polypeptides from serum. The beads may be conductive, forexample coated with a surface layer of gold, nickel, silver or otherelectrically conductive material. The beads may possess a variety ofmeasurable optical properties, e.g. color, fluorescence,phosphorescence, light scattering, reflectance, infrared and Ramanspectra, etc; such optical properties may be used to differentiateindividual beads in the bead-based analytical assays. Moreover, themeasurable optical properties of individual beads may be characteristicof the bead core, the bead surface or both. For example, fluorescentbeads may have a fluorescent core, in which the corresponding dye isdistributed throughout the bead core. Alternatively, fluorescent beadsmay have a fluorescent surface, in which the corresponding fluorescentdye is localized on or near the bead surface. Methods of makingcore-labeled and surface-labeled fluorescent and colored beads are knownin the art.

In an embodiment, the microbeads suitable for fabrication of themicroarrays disclosed in the present specification have sufficientlynarrow size distribution. The beads that have sufficiently narrow sizedistribution are often referred to as monodisperse beads and have a CV(coefficient of variation) at or below approximately 10%.

The microbeads suitable for fabrication of the microarrays disclosed inthe present specification may have various densities. In an embodiment,the beads have density below 0.5 g/cc (gram per centimeter cubed). Anexample of such beads is hollow glass microspheres. In an embodiment,the beads have density between 0.5 and 1 g/cc. An example of such beadsis polyethylene and polypropylene core beads that have density of about0.95-0.99 g/cc. In an embodiment, the beads have density between 1.0 and2.0 g/cc. An example of such beads is PMMA and cellulose acetate polymerbeads. In an embodiment, the beads have density greater than 2 g/cc. Anexample of such beads is glass and silica beads, includingpolymer-coated silica beads. In an embodiment, heavier beads, such asbeads having the density greater than approximately 1 g/cc, e.g. betweenapproximately 1.1 g/cc and approximately 2.0 g/cc may be easier todistribute into the microwells and retain on a microwell array plate.

In an embodiment, Janus microspheres and microparticles are used in themicroarrays disclosed in the present specification. A Janus particle isa particle that has a surface coating, which covers a fraction of itstotal surface, for example between 15 and 80% of the total surface,typically about 50% of the surface, e.g. a hemisphere or a half-shell. AJanus particle may have dual coatings and dual functionality. The dualfunctionality may include color, fluorescence, surface properties,surface reactivity, magnetic properties, electric properties etc.Custom-formulated Janus particles are available from several US-basedand overseas manufacturers of microspheres.

Microwell Array Plate

In an embodiment, a microwell array plate serves as the solid supportfor fabricating an array of microbeads. The microwell plates of theinstant disclosure may serve to retain individual beads in spatiallydistinct and addressable locations, may ensure that the beads remainreactive for an extended period of time, for example by preventingdegradation of the bead-conjugated active agents and prematuredissociation of the active agents from the beads, may enhance contactbetween a sample applied to the microarray and the individual reactivebeads, may enable facile release of analytes from the beads followingtheir reaction with the sample and subsequent localization of thereleased analytes in discrete and uniform spots, may enable detection ofanalytes by a specific analytical method and furthermore, may enablestorage and archiving of the reacted microarrays.

Various types of microwell array plates are suitable for fabricatingbead arrays according to the methods of the instant disclosure. Themicrowell plates may be obtained from commercial suppliers either as apre-fabricated item or as a custom-manufactured product. Suitablemicrowell plates may be also produced by a skilled person using methodsand devices that are known in the art. In particular, the plates may befabricated from various types of plain glass, chemically modified glass,photo-structured glass, micro-structured glass, fused silica, ceramics,polymers, metals, composite materials, thermoplastics, hydrogels andother suitable materials, for example fused fiber optic bundles. Themicrowell plates may include additional elements, such as a fiber-opticfaceplate. Depending upon the chosen material, various known techniquesof fabricating microwells or similar topological features, e.g. pits,depressions or indentations within the plate may be utilized thatinclude soft embossing, injection molding, laser ablation, acid etching,photolithography, soft lithography and others. The microwell plates mayhave variable linear dimensions, variable arrangement of individualmicrowells, variable shape and dimensions of individual microwells. Themicrowell plates may also incorporate various additional features, suchas microchannels or other microfluidic elements, various electroniccomponents including microelectrodes, optical barcodes, other forms ofbarcodes, RFID tags, labels, etc.

In an embodiment, there provided an additional single layer or severallayers deposited on the top surface of the microwell plate. Throughoutthis specification, the term “top surface” generally refers to thesurface of a microwell plate that contains openings into the microwells.The composition of the surface layer(s) may be selected to facilitatereaction between a sample introduced to the microarray and the reactivesites present within the microarray. The composition of the surfacelayer may be further selected to facilitate analysis of the reactedmicroarrays by one or several analytical methods. In an embodiment, thesurface layer is electrically conductive or at least static chargedissipative. The ability of the surface layer of the microwell plate todissipate static charge may facilitate application of specificanalytical methods, such as Matrix Assisted Laser Desorption IonizationMass Spectrometry (MALDI MS). It should be noted, however, that certainMALDI TOF MS instruments including AB SCIEX™ 4800 MALDI TOF/TOFANALYZER™ are configured such that they may readily analyze samplesdeposited on unmodified glass surfaces, which are normally notelectrically conductive. A sufficiently thin surface layer, for examplebetween 5 nm and 20 nm that contains a conductive metal oxide, such asIndium Tin Oxide (ITO), or alternatively a metal such as gold, chrome,nickel or similar may be used to modify a non-conductive surface of amicrowell plate to render it electrically conductive or at least chargedissipative while maintaining compatibility with various methods ofoptical detection, such as absorption, transmission and reflectionUV-visible spectroscopy, fluorescence and luminescence spectroscopy. Themetal-containing surface layer may be continuous or discontinuous: forexample a surface area of the microwell plate adjacent to an openinginto a microwell, e.g. an area within 1 μm, 5 μm, 10 μm, 25 μm, 50 μm orother distance from an opening into a microwell may not be covered bythe metal layer. The metal coating may be additionally applied to thebottom surface of individual microwells and/or the sidewalls ofindividual microwells. In an embodiment, the surface layer is opticallytransparent in the 360-800 nm wavelength range or in a smaller spectralregion within the 360-800 nm range, e.g. approximately 400-620 nm. In anembodiment, the surface layer has negligible auto-fluorescence.

The surface of the microwell plate may be hydrophilic, hydrophobic oromniphobic. In particular, a sufficiently hydrophobic or omniphobicsurface may help improve lateral resolution of the downstream microarrayanalysis by preventing excessive migration of analytes, which have beenreleased from the beads, on the microwell plate. The surface of themicrowell plate may exhibit sufficiently low non-specific binding, forexample low non-specific protein or peptide binding. In an embodiment,the surface of the microwell plate is chemically reactive, for exampleincludes a layer of an immobilized digestive enzyme such as trypsin, orother bioactive compound. In an embodiment, an immobilized bioactivecompound, such as the digestive enzyme, is localized on the sidewalls ofa microwell, on the bottom surface of a microwell, or both. The surfaceof the microwell plate may be further modified to make it suitable fordesorption-ionization of analytes using techniques known asDesorption-Ionization on Silicon (DIOS) and Nanostructured LaserDesorption Ionization (NALDI).

The surface of the microwell plate may have uniform surface propertiesthroughput an entire area of the plate. Alternatively, different regionsof the plate may have different surface properties. For example, theremay be provided a pattern of alternating hydrophobic and hydrophilicregions, so called “virtual wells”, that may help localize analytes,which have been released from individual beads, in discrete, compact anduniform spots. In general, as shown schematically in FIG. 3A, forcomposite microbead-microwell arrays comprising microbeads 302 submergedinto individual microwells on a microwell plate 301, the surfaceproperties may differ substantially in areas comprising an outer layerof a bead 311, areas surrounding openings into the microwells 314, areascomprising sidewalls of individual microwells 312 and areas comprisingbottom surface of individual microwells 313. In an embodiment, thesidewalls of individual microwells have greater surface roughness thanthe surface areas surrounding openings into the microwells. For example,the sidewalls of microwells fabricated by chemical etching, e.g. acidetching within quartz, glass or a ceramic substrate may have substantialsurface roughness. Quantitative methods of measuring surface roughnessmay be found in relevant publications (Degarmo, E. Paul; Black, J T.;Kohser, Ronald A. (2003), Materials and Processes in Manufacturing (9thed.), Wiley, ISBN 0-471-65653-4). Varying the surface roughness may beutilized to achieve or improve adhesion of individual beads to the innersurface of the microwells and thus enable immobilization of individualbeads within the microwells without forming a covalent bond between thebeads and the microwell plate. Known methods of surface modificationincluding sputtering, evaporation, chemical vapor deposition, chemicalsolution deposition, sol-gel technology and others may be also utilizedto alter the surface properties in a controlled fashion. The sputtering,evaporation and other related methods of surface modification may beutilized in combination with an appropriately designed mask toselectively modify specific areas within the microwell plate whileleaving the remaining areas unmodified. After performing the surfacemodification, various known methods of surface analysis includingoptical imaging, electron transmission microscopy and atomic forcemicroscopy (Pantano, P., and D. R. Walt. 1996. “Ordered nanowellarrays.” Chemistry of Materials 8:2832-2835) may be utilized to performdetailed characterization of the microwell plate surface.

In reference to FIG. 3B, it may be advantageous to provide a microwellarray plate 321, in which an individual microwell 322 has a diameter,which exceeds a diameter of the corresponding bead 323 by a certainvalue, e.g. by 10 μm, 25 μm, 50 μm, 100 μm or by other number. Onereason for providing microwells with the diameter greater than thediameter of the corresponding bead is to provide a suitable surface,such as the bottom microwell surface 324, which may perform one or morefunctions: (i) it may serve to localize analytes eluted from the bead (aprocess schematically depicted by an arrow 326), within a layer 325 and(ii) it may be accessible to a laser beam 328 of a mass spec instrument.It is noted that certain methods of mass spectrometry, such as MALDI,typically require hard, preferably conductive surfaces in order toefficiently desorb the analyte molecules and transfer them into the gasphase. Glass, metals and metal-coated surfaces are known examples ofsuitable solid supports for MALDI. In contrast, softer materials, suchas hydrogels, polymers and thermoplastics are less efficient solidsupports for MALDI. As a result, weak MALDI spectra may be recorded fromthe surface of a polymer bead, even if the bead has been coated with alayer of MALDI matrix. Therefore, eluting analytes from a polymer beadonto the suitable surface of a microwell plate may provide bettercompatibility with MALDI mass spectrometry because the analytes will bedesorbed from the surface of the microwell plate rather than from thebead. The bottom surface of an empty microwell is generally accessibleto the probing beam of a MALDI mass spec instrument, such as the UVlaser because the beam position is nearly orthogonal to the surface ofthe microwell plate in many MALDI MS instruments. In the case of abead-occupied microwell, a fraction of the bottom surface of themicrowell will be accessible to the probing beam, which will bedetermined by the difference between the microwell diameter and the beaddiameter. Many conventional MALDI TOF instruments feature laser beamwith a diameter between approximately 50 and 100 μm, however they canalso probe spots smaller than 50 μm in diameter by using a techniqueknown as oversampling.

It is noted that certain analyte desorption mass spectrometrictechniques, including SIMS, DESI and LAESI can analyze a variety ofsurfaces, including glass, polymers and plastics. Accordingly, elutionof analyte(s) from a bead onto the surface of a microwell plate may ormay not be required in assays involving the use of the abovementionedtechniques. In particular, for LAESI and related technologies it isusually necessary to provide a sample that contains sufficient amount ofwater, either in the liquid form or in the form of ice. This can beaccomplished by hydrating the bead array on a microwell array plate suchthat individual microwells will contain sufficient amount of water,which may be achieved by condensation of vapor on the microwell plate,by exposing the plate to a mist, by depositing nanodroplets in specificlocations throughout the plate or by spraying the plate with an aerosol.The analyte is eluted from a bead into a liquid-filled microwell, andthe contents of the microwell are then analyzed by mass spectrometry.Therefore, in an embodiment, a bead may occupy a larger portion of amicrowell in the SIMS, DESI and LAESI assays compared to the MALDIassays.

In reference to FIGS. 3C-3D, it is noted that because the MALDI processgenerally involves placing the sample into a high vacuum of a massspectrometer, beads manufactured from certain hydrogels includingagarose and cross-linked agarose may become desiccated and decrease insize (i.e. shrink) to a fraction of their original (i.e. hydrated) size.Desiccated beads may occupy a smaller portion of a microwell compared tothe hydrated beads and such effect should be taken into considerationwhen selecting the appropriate bead and microwell diameter. The decreasein the bead dimensions may be significant: desiccated agarose beads maybe reduced to about 25% or less of their original diameter andaccordingly, a greater portion of an area of the microwell may becomeexposed to the desorption-ionization laser beam of the massspectrometer. In an embodiment, when performing a MALDI assay it may beadvantageous to select a bead 332, such as an agarose bead, that has adiameter similar to the diameter of a microwell because the larger beadshave greater analyte binding capacity. Following the analyte releasefrom the bead and mixing the released analyte with MALDI matrix, thebead can be desiccated by air-drying, vacuum-drying or other similarmethods, which will cause the bead to shrink to a fraction of itsoriginal size 333. A microwell containing a desiccated bead along withthe analytes released from the bead will be generally analyzable byMALDI MS because the bead now occupies only a smaller fraction of thetotal volume of the microwell and does not interfere significantly withthe MALDI process. In an embodiment, the analyte is released from ahydrogel bead before the bead becomes desiccated. Some of the releasedanalyte may be localized on the bottom surface of a microwell, within alayer 335. Some of the released analyte may be localized on the topsurface of the microwell plate between openings into microwells, withina layer 336. In an embodiment, analytes localized on the bottom surfaceof a microwell and on the top surface of a microwell plate areanalyzable by MALDI MS. Other physico-chemical properties of the beadmaterial may be affected by the dehydration process as well (e.g. thebead material may become less soft), which may facilitate the use ofMALDI and other desorption ionization techniques.

In an embodiment, individual microwells within the microwell plate havesimilar shape and dimensions. For example, individual microwells may beshaped as cylinders of similar depth and diameter. In an embodiment, thedimensions of microwells are considered similar if they do not differ bymore than 30% between any two microwells within a microwell plate.Microwells of similar dimensions also have similar volume. Methods ofmanufacturing microwell plates featuring microwells of similardimensions are known. Furthermore, the use of various analyticaltechniques, such as optical and electron microscopy to verify dimensionsof the fabricated microwells is also known; an exemplary description canbe found in (Pantano, P., and D. R. Walt. 1996. “Ordered nanowellarrays.” Chemistry of Materials 8:2832-2835).

In an embodiment, microwell plates of the present disclosure have lineardimensions of approximately 25×75×1 mm, measured as width×length×height.Plates of the disclosed dimensions are size-compatible with numerousexisting optical imaging instruments, such as microarray scanners andfluorescent microscopes as well as many imaging-capable massspectrometers. In an embodiment, microwell plates have linear dimensionsof approximately 86×128 mm, measured as width×length to achieve sizecompatibility with numerous existing robotic systems designed forhandling 96-well, 384-well and 1536-well microtiter plates.

In an embodiment, the dimensions of individual microwells are selectedaccording to the dimensions of individual beads to be placed inside thecorresponding microwells. In an embodiment, the beads to be placedinside the corresponding microwells are approximately spherical andmonodisperse. In an embodiment, the cylindrical microwells selected foraccepting individual beads, which have a mean diameter of D, have depththat is not less than 0.3×D and not greater than 2.0×D. For example,microwells capable of accepting 90 micron beads should have depth notless than approximately 27 micron and not greater than approximately 180micron. In an embodiment, the cylindrical microwells for acceptingindividual beads, which have a mean diameter of D, have diameter that isnot less than 1.2×D and not greater than 2.5×D. For example, microwellscapable of accepting 90 micron beads should have diameter not less thanapproximately 108 micron and not greater than approximately 225 micron.Microwells featuring the disclosed dimensions may be capable ofretaining the suitably sized beads inside the microwells solely becauseof the spatial constraints imposed by the microwell shape and dimensionsand therefore may not require additional means of retaining beads on thesolid support, such as using an applied external magnetic field orforming a chemical linkage between the bead and a surface of themicrowell plate.

FIGS. 4A-4C schematically depict some shapes and dimensions ofindividual microwells, which may be utilized to secure position of abead on a microwell plate using spatial constraints imposed by themicrowell in which the bead is placed. The microwells featuring thedisclosed designs may be particularly useful when the beads are placedinside the microwells using centrifugation or an applied externalpressure, for example the beads initially positioned on a surface of amicrowell plate may be pushed into the microwells by a force applied tothe surface of the microwell plate. Furthermore, microwells featuringthe design similar to that depicted in FIGS. 4A through 4C may beparticularly useful when utilizing beads with a compressible core, suchas agarose beads or polystyrene beads. The internal diameter of themicrowells shown in FIGS. 4A through 4C varies along the depth of themicrowell and is larger near the opening into the microwell, i.e. nearthe top surface of the microwell plate. In an embodiment, the diameterof a microwell measured near the opening into the microwell is notgreater than 3-fold of the diameter of a bead to be placed inside themicrowell and not smaller than the diameter of the bead. In reference toFIG. 4A, a microwell has the shape of stacked cylinders with thecylinders of larger diameter placed near the opening into the microwell.In reference to FIG. 4B, a microwell has the shape of a truncatedinverted cone. In reference to FIG. 4C, a microwell has the shape of acylinder with an additional ring-like, or doughnut-shaped, or asimilarly functioning structure located below the opening into themicrowell, which serves to reduce the internal diameter of themicrowell. A bead that passes through the ring-like structure or astructure functionally similar to that shown in FIG. 4C may essentiallybecome “locked in” inside a microwell although it may still move aboutthe lower section of the microwell. The ability of a bead to move withinthe boundaries of a single microwell as opposed to being completelyimmobilized in a fixed position on a microwell plate may be advantageousbecause it may help improve the reaction kinetics between an activeagent bound to the bead and a sample present within a liquid mediumcontacting the bead. The limited movement of the bead within theboundaries of an individual microwell may be achieved, for example bymechanical agitation of the microwell plate using a vortexer, or by anapplied magnetic field for beads that comprise a material responsive toa magnetic field.

FIGS. 5A-5C schematically show beads placed inside the microwellssimilar to those depicted in FIGS. 4A-4C, respectively. In anembodiment, the beads may be placed into the microwells in a pre-swollenstate and allowed to swell after being placed inside the microwells.This may be achieved, for example by substituting the original liquidmedium with a solvent that causes the beads to swell. In an embodiment,the microbeads to be placed into the microwells depicted in FIGS. 4A-4Care either monodisperse or have sufficiently similar dimensions, forexample dimensions of any two microbeads within the bead library do notdiffer by more than 2-fold, preferably do not differ by more than1.5-fold, more preferably do not differ by more than 1.2-fold. Inreference to FIG. 5C, a group of arrows 501 schematically illustratesthe ability of a bead positioned inside a microwell to move within theboundaries of the microwell without escaping the microwell, as disclosedin greater detail in the previous paragraph. In an embodiment, beadmicroarrays featuring the microwell design similar to that shown inFIGS. 5A-5C may be utilized in conjunction with common laboratoryequipment designed to produce mechanical agitation, such as shakers,vortexers, nutators and similar. Numerous variations, alterations andmodifications of the disclosed microwell designs will be apparent to askilled person. For example, the generally flat sidewalls of acylinder-shaped microwell may additionally comprise one or several“spikes” 502 protruding from the sidewalls toward the interior of amicrowell and serving to immobilize a bead in a fixed position insidethe microwell, as schematically depicted as a side view and a top viewin FIGS. 5D and 5E, respectively. In an embodiment, the spikes 502 areshaped as needles measuring between approximately 5 to 90 micron inlength and approximately 5 to 50 micron in diameter and positioned at anapproximately 90 degree angle to the sidewall of a microwell, althoughthe latter parameter may vary. In an embodiment, the presence of spikesor similar topological features enables immobilization of a single beadwithin a relatively large microwell; that is a microwell whosedimensions and volume are considerably larger than the dimensions andthe volume of the corresponding bead. For example, using the designschematically depicted in FIGS. 5D and 5E or a similar design, a singlebead may be immobilized inside a microwell wherein the volume of themicrowell exceeds the volume of the immobilized bead by a factor of 2,3, 5, 10, 25 or even more. Importantly, the microwell plates featuringmicrowells of the disclosed design may be readily manufactured using themethods that are known and commonly practiced in the art. For example,the microwell plates may be fabricated from a photo-definable glass,which is glass that undergoes a phase transition (crystallization) uponexposure to electromagnetic radiation through a photomask followed byheating above the certain temperature. The exposed regions aresubsequently etched in a hydrofluoric acid at a rate much higher thanthe non-exposed regions thus enabling creation of a variety of customthree-dimensional features within the glass structure. An example of thephoto-definable glass is APEX™ glass available from Life BioScience Inc(Albuquerque, N. Mex.). Alternatively, microwells shaped similarly tothose depicted in FIGS. 4A-4C may be fabricated by photolithographyapplied to a thin film of polymer, such as the epoxy SU-8 photoresist.

In reference to FIG. 3A and also FIGS. 5A-5E, it can be seen that thereactive microbeads may be positioned inside their respective microwellssuch that a substantial fraction of the total surface of the bead willbe accessible to a sample contacted with the microarray. Increasing afraction of the total bead surface area, which is accessible to thesample, is advantageous because it increases the analytical sensitivityof an assay. In an embodiment, at least 30% of the total surface area ofa reactive microbead positioned inside a microwell is accessible to thesample, preferably at least 50% of the total surface area, morepreferably at least 75% of the total surface area, most preferably atleast 90% of the total surface area. If a bead is capable of movingwithin a microwell, as schematically illustrated in FIG. 5C, close to100% of the total bead surface area may be accessible to the sample.

In an embodiment, dimensions of the microwells are selected such thatthe beads are completely submerged into the wells and located at aspecific distance from the top surface of the microwell plate, forexample approximately 1, 3, 5, 10, 20, 50 or 100 micron below the topsurface. Placing the beads entirely within the microwells and providingan additional space between the bead and the top surface of themicrowell plate may help prevent or minimize evaporation of a liquidmedium surrounding the beads located inside the wells and thus helpmaintain the beads and the bead-conjugated active agents in a reactivestate. Importantly, placing the beads entirely within the microwellsalso enables, in an embodiment, spatially separating the subsequentchemical reactions performed on the microarray including reactions,which involve release of analytes from the reactive sites, as disclosedin greater detail elsewhere in this specification. In an embodiment, thearray of beads submerged inside the microwells additionally comprises aprotective cover, for example a removable plastic film or a thin glasscoverslip reversibly affixed to the top surface of the microwell plateand serving to further minimize the loss of the liquid mediumsurrounding the beads during the microarray shipping and storage. Suchprotective cover may also function to prevent other potentiallydeleterious effects affecting analytical performance of the microarrayincluding for example, accumulation of dust (fibers), photobleaching,oxidation, chemical contamination and bacterial contamination.

In an embodiment, dimensions of the microwells are selected such thatthe beads are only partially submerged into the wells and partially riseabove the top surface of the microwell plate, for example extendapproximately 1, 3, 5, 10, 20, 50 or 100 micron above the top surface ofthe plate.

In an embodiment, dimensions of the microwells enable positioning of nomore than one bead per microwell. However, microwell plates with largermicrowells may be also utilized, which will enable positioning of agreater number of beads, for example 2, 3, 5, 10 or more beads inside anindividual microwell. Microarrays featuring several distinct reactivebeads positioned inside a single microwell may provide an additionallevel of multiplexing, which is not available for microarrays featuringone bead per well. Such greater multiplexing capacity may beadvantageous in certain applications, however it should be ascertainedthat all individual beads within a microwell are accessible to thesample and can be decoded by an appropriate analytical method used toanalyze the microarray. In an embodiment, multiple identical, i.e.replicate beads are placed inside a single microwell. Identical orreplicate beads are conjugated to the same active agent and generallyhave very similar physical, optical and other properties. Microarraysfeaturing multiple identical beads per microwell may be useful if theamount of analyte from a single bead is not sufficient for analysis by adesired analytical method. Such microarrays may be particularly usefulin conjunction with the sample-consuming analytical techniques includingmass spectrometry.

Fabrication of a Reactive Bead Microarray

In an embodiment, the process of fabricating a microarray according tothe methods of the instant disclosure comprises the steps of providingbeads conjugated to one or more active agents and positioning the beadson a solid support in spatially distinct and addressable locations. Inan embodiment, multiple beads conjugated to distinct active agents maybe simultaneously placed on the solid support, such as a microwell arrayplate, to form a self-assembled array. For example, a suspension ofbeads in deionized water or in other suitable liquid medium may beplaced on a top surface of a microwell array plate and gently agitatedto help spread the beads across the plate surface. The beads will sinkinto the individual wells by gravity or as a result of subsequentlyapplied centrifugation, mechanical pressure or an external magneticfield. As little as ten or less and up to several thousand or evengreater than 1 million individual beads may be simultaneously arrayed ona solid support using the disclosed method. Providing microwell plateswith size-matching microwells will help ensure that only one beadoccupies a single well. Some wells may remain empty, i.e. not occupiedby a bead. Excess beads that remain on the surface may be subsequentlyremoved from the plate, for example by applying a stream of compressedgas to the plate surface, by gently rinsing the plate with a suitableliquid medium, e.g. deionized water, or simply by wiping the surfacewith a lint-free fabric, e.g. KIMWIPES™.

In an embodiment, the fabricated microarrays do not have positionalencoding. This may occur, for example, if the beads are randomlydistributed into individual microwells. In such case, the identity of abead-conjugated active agent may not be inferred from the location ofits carrier bead within a microarray. Accordingly, other methods ofmicroarray decoding or bead decoding that are known in the art may beimplemented. Non-limiting examples of such methods include: (i)identification of an active agent using optical spectroscopy bymeasuring optical properties of its corresponding carrier bead, opticalproperties of a label conjugated to the active agent or opticalproperties of the active agent itself; (ii) identification of an activeagent using mass spectrometry by measuring mass tags bound to itscorresponding carrier bead; the mass tags may be bound to the beadsurface or alternatively to the bead interior using the topologicallysegregated bilayer beads; (iii) identification of an active agent byprobing individual beads within the array with a decoding moiety, forexample a nucleic acid with a complementary sequence to a nucleic acidconjugated to a particular bead.

Identification of an active agent performed by measuring opticalproperties of its corresponding carrier bead is a particularly usefulapproach that may be readily implemented using the devices and methodsdisclosed in the instant specification, as well as the analyticalinstruments and protocols known in the field. Optically encodedmicrobeads are now widely used in numerous flow-cytometry basedanalytical applications including LUMINEX® assays, as well as planarbead array platforms, such as the one implemented in the LUMINEX®MAGPIX® instrument. Another method of optical encoding of the microbeadsrelies on utilizing rare earth elements, e.g. lanthanides and theircomplexes, which is commercialized in the PARALLUME™ analyticalmultiplexing platform. Briefly, one or several fluorescent dyes combinedat a specific ratio are incorporated into each bead to provide a uniqueoptical signature, which can be read using a flow cytometer, amicroarray scanner, or a fluorescent microscope. The fluorescenceemission wavelength and the relative intensity of signal at selectedwavelengths are measured and subsequently used to distinguish individualbeads and therefore the active agents conjugated to their respectivebeads. The process of fabricating a bead library featuring opticallyencoded microbeads normally comprises the steps of conjugating aselected active agent to one or more beads having an identical opticalsignature, recording the identity of the active agent conjugated to aspecific bead type and subsequently mixing the individual beadpopulations to create a multi-analyte assay panel. Due to the increasingpopularity of multiplexed suspension bead assays, it is now possible topurchase pre-fabricated peptide, protein and antibody bead librariesdesigned specifically for a particular bioanalytical assay. Such beadlibraries are sometimes referred to as bead sets, bead panels orsuspension bead arrays. It is also possible to purchase blank bead setsfeaturing optically encoded surface-activated beads that are ready to beconjugated to an active agent.

In an embodiment, dimensions and optical properties of the microwellplates, dimensions of individual microwells dispersed on the microwellplates, as well as the methods of microarray fabrication and the methodsof microarray analysis disclosed in the instant specification areselected to enable the use of commercially available optically encodedbead libraries, e.g. the bead kits available from LUMINEX® or itspartners “as is”, that is without making substantial modifications tothe beads or the bead surface chemistry. For example, the microwellplates may be fabricated from fused fiber optic bundles, fromphoto-structured glass or from fused silica to contain microwells, whichare sized to accept the approximately 6 micron diameter standard-sizemicrobeads utilized in the flow cytometry-based LUMINEX® assays.Importantly, the amount of analyte released from even such small beadsmay be sufficient for the downstream analysis by mass spectrometry,which often requires sub-femtomole amounts of analyte for detection. Inan embodiment, the microwell plates are compatible with fluorescencemeasurement of beads that are optically encoded by a combination of rareearth elements, for example the optically encoded beads that arecurrently commercially available and marketed under the trademarkPARALLUME™ by Parallel Synthesis Technologies Inc of Santa Clara, Calif.Such bead compositions, also disclosed in the U.S. patent applicationSer. No. 12/091,900, now U.S. Pat. No. 8,673,107 are capable ofgenerating thousands to millions of distinguishable optical codes. Inthe context of this specification, the term “fluorescence measurement ofa bead” and similar terms may refer to the ability to perform excitationof one or more fluorescent dyes localized on an individual bead and tosubsequently record emission spectra from the excited fluorescent dyeslocalized on the bead wherein the fluorescence spectra are recorded at asingle wavelength or at multiple wavelengths. The abovementioned abilityto acquire fluorescence spectral data from a single bead may alsoinclude the ability to perform quantitative measurement, e.g. to measurethe emission signal intensity as a function of the dye concentration onits carrier bead. In an embodiment, two distinct fluorescent dyeslocalized on the same bead may have similar excitation spectra butdistinct emission spectra. Such dyes may be simultaneously excited by asingle excitation source but their emission spectra may be measured attwo different wavelengths, either concurrently by utilizing two distinctdetectors tuned to the corresponding wavelengths, or consecutively by asingle detector at each wavelength individually. Methods of readingoptical bead signatures in the planar bead array format are known in theart, for example they are disclosed in the U.S. patent application Ser.No. 12/517,248, now U.S. Pat. RE44,693 and U.S. patent application Ser.No. 12/091,900, now U.S. Pat. No. 8,673,107, among other references.

Importantly, as disclosed in this specification and also in the U.S.patent application Ser. No. 13/369,939, Publication No. 2012-0202709 A1,the entirety of which is incorporated herein by reference, optical, e.g.fluorescent spectra of beads arrayed on a microwell plate may berecorded not only from the bottom of microwells, for example via opticfibers, which functionally connect individual microwells to an opticaldetector, but also from the top surface of a microwell plate, e.g. viaopenings into the microwells when the beads are either partially orentirely submerged into the microwells. In the latter configuration,beads arrayed on a microwell plate may be directly measured by opticalspectroscopy in a specific wavelength range even though the material ofthe microwell plate itself may not be compatible with such measurement.For example the PARALLUME™ beads normally require excitation byultraviolet radiation near 325 nm provided by a He—Cd laser. On theother hand, some commonly used fluorescent dyes emit in the near-IR partof the spectrum. Consequently, many materials, e.g. certain types ofglass that are opaque in the near-UV or near-IR spectral regions maystill be used for the measurement of beads encoded by dyes withexcitation or emission in these spectral regions as long as the opticalreadout is performed directly from the beads arrayed near the topsurface of the microwell plate or via openings into the microwells.

There exist various types of solid supports suitable for fabricatingmicroarrays of the present disclosure, which are compatible with themethods of optical imaging such as fluorescence imaging, luminescenceimaging and colorimetric imaging. In particular microwell platesfabricated from fiber optic bundles and many types of microwell platesfabricated from quartz, from fused silica or from photo-structured glassare fully compatible with the optical detection methods. Besides glass,certain thermoplastics including for example polystyrene, polypropylene,polycarbonate, cyclic olefin copolymer (COC), cyclic olefin polymer(COP), acrylic (PMMA) and thermoplastic elastomers possess desirableoptical characteristics, namely negligible autofluorescence (preferablyfor the fluorescence excitation throughout the 250-700 nm wavelengthrange and fluorescence emission throughout the 300-850 nm wavelengthrange; in particular for the common fluorescence excitation channelscentered around 405 nm, 488 nm, 532 nm, 576 nm and 635 nm) andtransparency in the visible range (preferably for the entire 250-850 nmwavelength range, although smaller spectral range may be acceptable; theoptical transmission in this spectral range should be over 50%,preferably over 75%, more preferably over 90%). Accordingly, themicroarrays of the present disclosure that are fabricated from opticallyencoded bead libraries will be readily decodable by optical imaging aslong as the solid support, on which the beads are arrayed, is compatiblewith the optical imaging. In an embodiment, the bead microarray decodingprocedure is performed on an unreacted microarray, i.e. prior toexposing the microarray to a sample. In an embodiment, the beadmicroarray decoding procedure is performed on a reacted microarray, i.e.subsequent to exposing the microarray to a sample. In an embodiment, themicroarray decoding procedure is performed by the microarraymanufacturer prior to shipping the microarray to a customer. In anembodiment, the microarray decoding procedure is performed by thecustomer, i.e. the end-user using the customer's own optical imaginginstrument, e.g. a microarray scanner and the list of optical bead codesprovided by the microarray manufacturer. Potential benefits associatedwith having the microarray decoding procedure performed by themicroarray manufacturer rather than by the end-user may include one ormore of the following: (i) elimination of possible errors arising fromthe customer's interpretation of the measured optical bead codes and norequirement for the customer to have access to an optical scanner, forexample if the reacted microarray is only analyzed by mass spectrometry;(ii) the ability to store the functional fabricated microarrays for anextended period of time even if the optical bead labels are unstable orsusceptible to degradation, for example due to oxidation orphotobleaching; (iii) the ability of the microarray manufacturer toutilize custom-developed, proprietary or non-standard optical beadlabels.

In an embodiment, mass tag-based bead encoding techniques are utilized.The mass tags may be conjugated to the outer layer of a bead;alternatively the mass tags may be conjugated to the inner layer locatedinside a topologically segregated bilayer bead. Localization of masstags within the bead interior may be advantageous because it minimizesor eliminates the possibility of a cross-reaction between the mass tagand the sample contacted with the bead microarray. The U.S. patentapplication Ser. No. 13/172,164, Publication No. US 2012-0077688, theentirety of which is incorporated herein by reference, discloses severalexemplary methods of fabricating mass tag-encoded reactive beads, forexample mass tag-encoded beads conjugated to a capture agent, such asantibody. The U.S. patent application Ser. No. 13/369,939, PublicationNo. US 2012-0202709 A1, the entirety of which is incorporated herein byreference, discloses several methods for sequentially releasing multipleanalytes from a single bead and localizing the released analytes in asingle spot on the solid support.

The instant specification discloses various methods of producing, usingand analyzing individual beads and bead libraries, in which a bead isreversibly conjugated to a plurality of oligomer molecules, such aspeptides, peptidomimetics, oligonucleotides, peptide nucleic acids(PNAs), carbohydrates, etc. For example, a single bead may contain about1 nmole of a conjugated peptide. In an embodiment, such oligomermolecules are synthesized on the bead and remain conjugated to the beaduntil the bead is ready to be analyzed by mass spectrometry, e.g. untilthe bead is placed on a microwell array plate. A non-limiting example ofa compound synthesis on beads is the Fmoc chemistry-mediated solid phasesynthesis of peptides on TENTAGEL™ beads. In an embodiment, the oligomermolecules are conjugated to the bead via a labile linker, e.g. aphoto-labile, acid-labile or thermo-labile linker. In an embodiment, thelinker is incorporated during the on-bead synthesis. It is noted thatthe oligomer molecules synthesized on beads may be used in thedownstream analytical applications without further enrichment orpurification, i.e. using the “on-bead” purity.

In the context of this specification, the term “purity” may refer to thechemical purity of a compound as determined by an appropriate analyticalassay. For example, high performance liquid chromatography (HPLC) is atechnique, which is frequently used to measure the purity of variouscompounds. Alternatively, a spectrographic method such as massspectrometry may be used to measure the compound purity. The compoundpurity may be measured and reported as a percentage value, which mayrefer to the amount of a desired analyte (e.g. a peptide with thecorrect amino acid sequence) relative to the amount of a total analytein a sample (e.g. including partially synthesized peptides, peptideswith partially cleaved de-protecting groups, prematurely terminatedpeptides etc). The peptide and other manufacturers frequently specifythe product purity in percentage points e.g. 50%, 75%, 95%, 99%. In somecases, the product purity is estimated rather then directly measured,for example when the synthesis process is well-known, which may includeknown reaction conditions, known precursors, known source of theprecursors, known synthetic scale, etc. In such case, the purity of thefinal product may be given within a certain range, e.g. greater than 50%pure or 50% to 75% pure.

The instant specification discloses several embodiment methods of usingthe compounds synthesized on beads, including their use as an activeagent, e.g. an enzyme substrate or a capture agent, or alternatively asa molecular weight tag (a “mass tag”). In the latter example, theon-bead synthesized compound serves to provide identification of aparticular bead, This is achieved by releasing the mass tag from thebead, measuring its molecular weight by mass spectrometry and matchingthe measured molecular weight to a particular mass tag and therefore, toa particular bead. The mass-tag conjugated bead may be also separatelyconjugated to an active agent, e.g. an antibody, a protein or a ligand.Alternatively, the mass-tag conjugated bead may feature specificchemistry, which will enable conjugation of an active agent to the beadin the future. Examples of the specific chemistry include carboxyl andamino-reactive beads, NHS-reactive beads, epoxy beads, aldehyde beads,protein A, protein G and protein L conjugated beads,Streptavidin-conjugated beads, biotin-conjugated beads, Ni²⁺-conjugatedbeads, Co²⁺-conjugated beads, EDTA-conjugated beads, anti-His tagantibody conjugated beads, anti-FLAG™ antibody conjugated beads,anti-human IgG antibody conjugated beads, glutathione-conjugated beads,etc.

Examples may be found in the prior art, which utilize bead mass tags foridentification of beads by mass spectrometry. However, these earlierexamples rely on sufficiently pure compounds, e.g. the compounds thathave approximately 90% purity or greater. As a result, only a singlesignal in a mass spectrum may be recorded from such mass tags, which isnormally an isotopic envelope consisting of several peaks spaced apartby approximately 1 Da due to the presence of a ¹³C isotope within themass tag sequence. Furthermore, the mass tags are usually firstsynthesized, then modified and subsequently bound to the beads usingmulti-step, labor-intensive and cumbersome procedures and expensivereagents, e g. NEUTRAVIDIN™ and photolabile biotin.

In contrast, in an embodiment the instant specification discloses thefabrication, use and analysis of mass-tag conjugated beads, in which themass tags are synthesized directly on the beads with a cleavable linkerincorporated during the mass tag synthesis. Furthermore, the instantspecification discloses mass tags capable of generating at least twodistinct signals in the mass spectra, which are spaced apart by at leastseveral Da. In an embodiment, one of these signals corresponds to thecorrect (predicted) molecular weight of the mass tag, while theadditional signals correspond to molecular weights of the by-productsgenerated during the mass tag synthesis reaction.

The EXAMPLES section of the specification discloses severalbead-conjugated mass tags, which when released from the beads andmeasured by mass spectrometry generate multiple peaks in the massspectra. Such mass tags are advantageous because instead of a singlepeak (or a single isotopic envelope), a series of two, three or morepeaks may be observed in the mass spectrum, providing a unique spectralprofile (“mass tag signature”) suitable for unambiguous identificationof a specific mass tag within a library comprising multiple mass tags.In addition to the more facile mass tag identification, synthesizingcompounds directly on beads with the “on-bead” purity is potentiallyfaster and cheaper compared to individually conjugating highly purifiedcompounds to surface-activated beads.

In an embodiment, the microarrays of the instant disclosure arefabricated such that the position of each individual bead within themicroarray is known and recorded for the purpose of subsequent decodingof the microarray. Such bead microarrays possess positional encoding.Bead microarrays featuring positional encoding may be fabricated usingvarious methods. For example, the populations of beads comprisingdifferent active agents may be sequentially deposited at specific,pre-determined locations within the microarray either one at a time orseveral at a time using a single-channel pipette, a multi-channelpipette or other similarly functioning bead dispensing device, eithermanually or in automated fashion. An example of an automated devicecapable of dispensing beads into wells of a microwell plate is COPAS™available from Union Biometrica (Holliston, Mass.). The disclosed methodis conceptually similar to the method of fabricating conventional planarmicroarrays that involves robotic printing of individual reactive spotson a chemically activated surface of the solid support. In the methodsof the present disclosure, individual beads to be arrayed on a microwellplate are preferably contacted with the microwell plate at specificpoints determined by geometrical parameters or layout of the grid ofmicrowells, such that a bead is released from the bead-dispensing deviceeither near an opening into a microwell or directly into an opening intoa microwell. As a result, individual beads contacted with the solidsupport using the disclosed procedure will promptly sink into theircorresponding microwells thus minimizing the risk of a random beadmigration across the surface of the microwell plate. To facilitate theprompt transfer of beads from a bead-dispensing device to a solidsupport, the microwells may be pre-filled with a liquid medium that issimilar in composition to the medium in which the beads are supplied.

The beads arrayed on a solid support do not necessarily form a covalentchemical bond with the solid support. As disclosed previously, thespatial constraints imposed by the dimensions of individual microwellswithin a microwell array plate may be sufficiently rigid to enableretention of beads within their corresponding microwells even withoutforming a covalent linkage between a bead and the solid support. In theabsence of covalent bonding, the beads may be immobilized via othertypes of physico-chemical interactions such as electrostaticinteractions, van der Waals interactions, hydrophobic interactions,hydrogen bonding etc. It is also possible to deposit small amounts of anadhesive, such as MOWIOL® or Aqua-Poly/Mount in specific locationsthroughout the solid support to create an array of sticky spots, whichwill function to capture and retain individual beads on the solidsupport. If a mass spectrometric method, such as MALDI TOF MS is usedfor the subsequent analysis of the fabricated microarray, the adhesivespots may be subject to additional requirements such as: (1) theadhesive material should be sufficiently resistant to organic solventsutilized in the MALDI sample preparation workflow; (2) the adhesivematerial should not be detectable in the measured mass spectra or shouldgenerate relatively weak signal that will not affect identification ofthe analytes present on the microarray and (3) the adhesive materialshould not prevent desorption of the analytes from the solid support. Inan embodiment, magnetic beads may be retained on the microwell plate byapplying an external magnetic field. In an embodiment, a permeablemembrane or a porous film is affixed to the top surface of the microwellplate to retain individual beads inside their corresponding microwells.The methods of retaining beads on the solid support that do not utilizethe surface chemistry of the solid support may be particularlyadvantageous because the surface of the microchips may be accordinglymodified to perform other useful functions, for example it may comprisean immobilized digestive compound, e.g. trypsin, or a layer ofenergy-absorbing matrix for nanostructure-initiator mass spectrometry ora layer of material with specific optical properties, which will providecompatibility with a fluorescence- or luminescence-based assay readout.Alternatively, the surface of the solid support may be modified toenable retention of the analytes, which are released from the beads, inthe vicinity of their respective beads, for example via hydrophobicinteractions. Overall, various methods of attaching beads to the solidsupport including covalent bonding may be found in the prior art, forexample U.S. Pat. No. 6,429,027, which discloses arrays of microspheres,and U.S. Pat. No. 5,356,751, which discloses arrays of tacky areas.Importantly, the known methods of attaching beads to the solid supportmay be implemented in combination with various microwell array platesdisclosed in the instant specification; for example either a bottomsurface of a microwell or sidewalls of a microwell may be modified withan adhesive material for the purpose of binding and retaining a beadinside the microwell.

In an embodiment, a bead is tethered on the microwell plate by acovalent linkage formed between the surface of the microwell plate andthe compounds linked to the bead. For example, the microwell plates ofthe instant disclosure may be surface-modified to produce epoxy,aldehyde or NETS-activated surfaces, as well as several other reactivesurfaces. A protein-conjugated bead or a polypeptide-conjugated beadplaced onto such epoxy, aldehyde or NHS-activated surface, will readilyform a covalent linkage between a primary amine group within thebead-conjugated polypeptide and the surface of the solid support, whichwill serve to tether the bead to a specific location inside a microwell.

In an embodiment, a bead microarray featuring positional encoding isfabricated by simultaneously contacting multiple identical beads with asolid support, e.g. a microwell plate, such that the beads are placedinto microwells, which are adjacent to each other and located in apre-determined area of the solid support. This approach allowssimultaneous arraying of multiple replicate beads and even though theposition of each individual bead is determined randomly, the array stillhas positional encoding because every bead placed within thepre-determined area carries an identical active agent. Furthermore,known parameters of the microwell array grid, e.g. the diameter of wellsand the well-to-well spacing may be utilized to determine preciseposition of each bead on the solid support. By way of a non-limitingexample, over 300 microwells may be simultaneously filled with beads atone bead per well occupancy by loading a suspension of 34 μm diameteragarose beads into a 1 mm internal diameter pipette tip, subsequentlycontacting the pipette tip with the surface of a microchip featuring ahexagonal array of microwells that are 42 μm wide and have 50 μmwell-to-well spacing, releasing the beads from the pipette tip andallowing the beads to sink into the microwells.

In an embodiment, a bead microarray featuring positional encoding isfabricated using a combination of a microwell array plate and a gasket,which is reversibly attached to the top surface of the microwell arrayplate. The gasket serves to subdivide the microwell plate into distinctregions during the bead deposition process, for example the number ofdistinct regions within a single microwell plate may be 4, 16, 32, 64 orother number. Individual beads located within a single region may beconjugated to the same active agent. Alternatively, the beads may beconjugated to distinct active agents.

For bead microarrays fabricated on microwell array plates it may bedesirable to achieve close to 100% well occupancy so that almost everymicrowell is occupied by a bead. Achieving close to 100% well occupancyallows the highest possible density of the reactive sites on amicroarray and may facilitate the downstream microarray readout andanalysis of the acquired data. In an embodiment, more than 75% ofmicrowells of a microwell plate contain a bead. In an embodiment, morethan 90% of microwells of a microwell plate contain a bead. In anembodiment, more than 95% of microwells of a microwell plate contain abead. Of course microarrays featuring less than 100% microwell occupancyare also functional. In an embodiment, some microwells contain two ormore beads, although the number of such microwells is preferably limitedto 5% of the total number of microwells or less, more preferably to 1%or less. Lastly, it is possible that a small number of beads may bestuck on the surface of a microwell plate outside the microwells. Thesepossibilities should be taken into account when the microarray data isanalyzed, for example in the microarray grid alignment and microarraysegmentation procedures.

In an embodiment the microarrays of the instant disclosure may possessno conventional means of encoding the active agent. Such microarrays aresometimes referred to as encoder-less microarrays. In an embodiment, theidentity of an active agent conjugated to a bead may not be determinedfrom a position of the bead within the microarray, or from optical orother relevant properties (e.g. diameter) of the bead. In an embodiment,the identity of an active agent conjugated to a bead may not bedetermined by binding a probe, such as a decoding ligand to the bead. Inan embodiment, an active agent is conjugated to a bead that does nothave a mass tag. An example of such encoder-less microarray is a randombead array comprising optically indistinguishable beads individuallyconjugated to different active agents. Another example is a one bead-onecompound (OBOC) combinatorial bead library comprising hundreds,thousands or even millions of distinct beads randomly arrayed on amicrowell plate. Encoder-less bead microarrays may be also fabricatedfrom bead libraries produced by emulsion-based chemical reactions.Consequently, an active agent may serve as its own code and themicroarray decoding procedure may comprise releasing the active agentfrom its corresponding bead followed by identification of the activeagent using mass spectrometry. The active agent may be released from itscarrier bead either as an intact molecule, or as one or severalmolecular fragments produced for example, by enzymatic digestion.Identification on an active agent by mass spectrometry may comprisemeasurement of its molecular weight, as well as measurement of molecularfragments generated by fragmentation mechanisms known as PSD, ETD, CID,etc. More than one mass spectrum may be acquired from the same locationin order to identify a specific active agent, for example spectra may beacquired in a different m/z range, or in a different mode, e.g. linear,reflector and MS-MS using different precursor ions.

In an embodiment, multiple distinct active agents are conjugated to asingle bead. In an embodiment, at least 2 distinct active agents areconjugated to a single bead. In an embodiment, the number of distinctactive agents conjugated to a single bead is between 2 and 20. In anembodiment, the number of distinct active agents conjugated to a singlebead is between 20 and 100. In an embodiment, the number of distinctactive agents conjugated to a single bead is greater than 100. In anembodiment, each of the distinct active agents conjugated to the samebead is releasable from the bead and analyzable at least by massspectrometry. In an embodiment, distinct active agents conjugated to thesame bead are conjugated by an identical linker. In an embodiment, atleast some distinct active agents conjugated to the same bead areconjugated by different linkers, for example different photolabilelinkers, which are cleavable by light of different wavelengths. In anembodiment, each of the distinct active agents conjugated to the samebead is present on the bead in at least 10⁶ copies, preferably in atleast 10⁹ copies, more preferably in at least 10¹⁰ copies. In anembodiment, each of the distinct active agents conjugated to the samebead is present on the bead in the amount that is sufficient for therelease of at least 1 femtomole of analyte from the bead for thesubsequent analysis by mass spectrometry, preferably at least 10femtomoles, more preferably at least 100 femtomoles. A non-limitingexample of multiple distinct agents conjugated to a single bead is adegenerate peptide library; various types of those are known in the artand some are described in the Experimental Examples.

Some of the options of the microarray decoding and the reaction readout,which may be utilized in conjunction with the composite microarraysdisclosed in the instant specification, are listed in Table 1 shown inFIG. 6. Various additional readout options, for example the beadmicroarray decoding using radio frequency (RFID) tags will be apparentto a person skilled in the art.

The composite bead-based microarrays disclosed in the instantspecification are compatible with various methods of storage andshipping of biological materials, which may help ensure stability of theactive agents conjugated to the beads. For example, the bead microarraysmay additionally include bacterial growth inhibitors such as sodiumazide, protease and nuclease inhibitors, DTT, or glycerol for storagebelow −18° C. Furthermore, the bead-based microarrays may be stored in alight-blocking container, particularly when the active agents compriselight-sensitive compounds.

In an embodiment, the disclosed methods of fabricating bead-basedmicroarrays enable “on demand” assembly of a microarray from the stocklibraries of microbeads conjugated to different active agents. Suchprocess may include the steps of combining aliquots of beads fromdifferent bead stock libraries and positioning the beads on a microwellarray plate. In addition to selecting a particular type of an activeagent, an appropriate number of replicate beads, e.g. approximately 2,10, 100 or a greater number may be selected for each active agent. In anembodiment, such process is performed using the method offluorescence-activated cell sorting (FACS). An example of an instrumentcapable of sorting beads on the basis of their fluorescence propertiesis COPAS™, which is available from Union Biometrica (Holliston, Mass.).Bead-dispensing robotic devices are also available from othermanufacturers, e.g. Digilab (Marlborough, Mass.). The microarray mayalso comprise areas containing unoccupied, i.e. empty wells, which willenable the end-user to add their own microbeads to a pre-fabricatedmicroarray.

In an embodiment, surface-activated microbeads, for exampleCNBr-activated or NETS-activated agarose beads available from ThermoScientific and GE Healthcare Life Sciences or succinimidyl esterTENTAGEL® beads available from Rapp Polymere may be arrayed on amicrowell plate using the previously disclosed methods to create anarray of surface-activated sites capable of binding a particular activeagent, e.g. an antibody. Different active agents may be subsequentlydispensed at specific locations within the microwell plate and reactwith the surface-activated microbeads positioned inside the microwellsin order to create a reactive bead microarray featuring positionalencoding. The experimental methods of binding active agents to thesurface-activated microbeads, which are arrayed on a microwell plate,are generally similar to the conventional methods of immobilizingcompounds on surface-activated microbeads suspended in a liquid medium.In particular, detailed experimental protocols are readily availablefrom the manufacturers of surface-activated beads including ThermoScientific and GE Healthcare Life Sciences, among numerous othersources.

In an embodiment, there disclosed a kit for performing bead-basedmultiplexed reactions with the downstream analytical readout by massspectrometry and optionally by fluorescence. In an embodiment, the kitconsists of one or several microwell array plates and a pre-determinedamount of size-matching microbeads suitable for fabrication of a beadarray on the microwell array plate at one bead per microwell occupancy.The microbeads may be supplied in a single container, e.g. EPPENDORF®microcentrifuge tube or in several containers. The number of aliquotedbeads in each container may be approximately equal to the number ofmicrowells on a microwell array plate or a section thereof. In anembodiment, the number of beads is between 50% and 75% of the number ofmicrowells on a microwell array plate or a section thereof.

Reacting a Microarray with a Sample

Several embodiment methods of reacting a bead microarray of the instantdisclosure with a sample are schematically depicted in FIGS. 7A-7F anddescribed in greater detail below.

In general, various types of biochemical reactions may be probed usingthe microarrays of the instant disclosure including reactions that areamenable to screening using conventional printed microarrays andsuspension bead assays. For example, the microarrays of the instantdisclosure may be utilized to measure enzymatic activity of the sample,in such case the sample may comprise a purified enzyme, a mixture ofenzymes or a biological medium suspected of possessing an enzymaticactivity, e.g. a cell lysate.

Alternatively, the microarrays of the instant disclosure may be utilizedto detect the presence of a particular analyte in the sample: thedetection may comprise qualitative detection of the analyte,quantitative detection of the analyte or detection of the presence ofthe analyte above or below a certain threshold.

Alternatively, the microarrays of the instant disclosure may be utilizedto detect binding of a particular compound, such as a drug candidate, toits intended target, such as a protein or a protein complex.

Biochemical reactions probed by the microarrays of the instantdisclosure may comprise an affinity binding reaction, such asantibody-antigen interaction, receptor-ligand interaction,lectin-polysaccharide interaction, enzyme-drug interaction, binding of acomplementary DNA or RNA sequence, etc. A reaction comprising binding ofan antigen to a bead-conjugated antibody is known in the art as affinityimmunoprecipitation (IP) or affinity immuno-precipitation reaction. Areaction comprising an affinity binding, which involves a proteincomplex, is known as co-immunoprecipitation (co-IP). A reaction in whichthe IP technique is used to separate analytes on individual beads isknown as immunoaffinity purification (IAP). The abovementioned types ofreactions may be implemented on the microarrays of the instantdisclosure.

In an embodiment, a microarray of the instant disclosure may be utilizedto probe reactions, which comprise release of analyte(s) from anindividual reactive site of the microarray. The analyte release mayoccur, for example from contacting the microarray with a samplecontaining a digestive compound or a competitive binding ligand. Anotherexample of an analyte release reaction is time-dependent diffusion of ananalyte out of the reactive site on a microarray, which in this case maycomprise an ion-exchange resin or alternatively a topologicallysegregated bilayer bead. Yet another example of an analyte releasereaction is dissociation of an antigen from its respective antibody orfrom an aptamer; a further example of an analyte release reaction isrelease of a drug from its solid phase carrier, for example adrug-conjugated micro- or nanoparticle. Another example of an analyterelease reaction is release of an analyte initially encapsulated insidea micro- or a nanoparticle made of a polymer, such aspoly(lactic-co-glycolic) acid or PLGA, which undergoes hydrolysis uponcontact with water or another solvent. The analyte release reaction mayalso comprise photolysis of a light-sensitive chemical bond between theanalyte and the reactive site.

In an embodiment, the microarrays of the instant disclosure are used forscreening and functional characterization of linkers and/or spacers thatprovide conjugation of an active agent to a bead. For example, an activeagent may be conjugated to individual beads via different linkers, e.g.linkers that have different length, different chemical structure,different reactivity toward a particular digestive compound, etc. It isknown that changing the linker structure may either improve or worsenthe active agent “presentation” on the bead, that is the ability of theactive agent to react with a sample contacting the bead. One examplewhere such effect has been previously observed is a chemical reactionbetween a bead-conjugated peptide and a sample containing the proteinkinase capable of phosphorylating the bead-conjugated peptide. It hadbeen observed that by varying the length and chemical structure of thepeptide-bead linker, it was possible to significantly improve thereactivity of the bead-conjugated peptide toward the enzyme, as measuredby the extent of the peptide phosphorylation on bead. Accordingly, it ispossible to provide a library of identical active agents conjugated totheir respective beads via linkers of different structure. Such librarymay be subsequently used to perform functional, e.g. enzymatic screeningin a bead microarray format followed by analysis of the reacted beads bymass spectrometry and/or optical spectroscopy. In an embodiment, thebead library containing the different linkers is fabricated using themethods of combinatorial synthesis, for example the one bead onecompound (OBOC) or one bead two compound (OB2C) methods. Note that theexperimental methods disclosed in the instant specification enablerelease of the linkers from their corresponding beads for the subsequentidentification and/or characterization by mass spectrometry.

In an embodiment, the microarrays of the instant disclosure are used forother forms of functional characterization of linkers and/or spacersthat provide conjugation of an active agent to a bead. In contrast tothe functional assays disclosed in the preceding paragraph, theindividual linkers and/or spacers may be assayed to determine theirefficiency in the reactions involving the release of an active agentfrom a bead. For example, the release reaction may involve photolysisreaction, hydrolysis reaction, enzyme-catalyzed hydrolysis reaction,etc. Accordingly, a bead library containing an analyte conjugated to thebeads via linkers of different structure may be fabricated and screenedin a bead array format to determine the efficiency of the analyterelease reaction, which may include characterization of the reactionkinetics.

In an embodiment, the analyte released from a reactive site of amicroarray is localized on the microarray in the vicinity of itsrespective reactive site. In an embodiment, the analyte released from areactive site of a microarray is localized within 250 μm (250 micron)from the respective reactive site. For example, analyte released from a300 micron diameter carrier bead, which is positioned inside a 500micron wide, 500 micron deep cylindrical microwell, may be predominantlylocalized within a 250 micron distance from its carrier bead. In anembodiment, the analyte released from a reactive site of a microarray islocalized within 100 μm (100 micron) from the respective reactive site.In an embodiment, the analyte released from a reactive site of amicroarray is localized within 30 μm (30 micron) from the respectivereactive site.

More than one analyte release reaction may be performed concurrently orsequentially in the same reactive site within the microarray.Furthermore, more than one analyte binding and subsequent analyterelease reaction may be performed concurrently or sequentially in thesame reactive site within the microarray. Such “capture and release”microarrays provide substantial advantages over the conventionalmicroarrays, which are usually not configured for releasing the analytesfrom their reactive sites.

In an embodiment, an exemplary microarray of the present disclosurecomprises a microwell array plate 701 and a plurality of microbeads 704placed into individual microwells 702, as schematically depicted in FIG.7A. In reference to FIG. 7B, a microbead may comprise a single activeagent or several distinct active agents 712 conjugated to the beadsurface 710 (or the bead interior) via an optional linker 714. In anembodiment, a microbead conjugated to an active agent and placed into amicrowell functions as a reactive site of a microarray.

In reference to FIG. 7C, a microarray reaction may comprise contactingthe microarray 721 with a liquid sample 724, which may be a solution, acolloid, a suspension, an emulsion or an aerosol. The sample 724 may besimultaneously contacting multiple reactive sites 722 such that theindividual reactive sites 722 are fluidically connected, i.e. an analytemay migrate between adjacent or non-adjacent reactive sites within themicroarray. The disclosed method is conceptually similar to theconventional methods of reacting a printed microarray or a suspensionbead array with a sample, in which the sample is introduced in a singlealiquot. In practice, this may be accomplished by contacting asufficiently large amount of the sample, e.g. 100 μL, 500 μL or 1 mLvolume of a liquid medium containing the sample with the microarray andallowing the sample to spread on the microarray. Following the reactionbetween the microarray and the sample, the unreacted andnon-specifically bound compounds may be removed by rinsing themicroarray with an appropriate medium, e.g. a buffer, a mild detergentor deionized water.

In reference to FIG. 7D, a microarray reaction may comprise contactingthe microarray 731 with a liquid sample 734 such that the individualreactive sites 732 on the microarray are fluidically disconnected fromeach other. In this approach, each sample or a fraction of the samplecontacts only one reactive site on the microarray and the analytemigration between the different reactive sites does not occur. This maybe accomplished, for example, by performing the microarray reactionentirely within individual microwells or alternatively by formingsufficiently small sample-containing droplets around the individualreactive sites on the microarray. In this approach, the sample may becontacted with the microarray reactive sites by using a nebulizer, a TLCsprayer, a liquid microdispensing robot or other similarly functioningdevice. In an embodiment, the sample solution may be applied to thesurface of a microwell plate in bulk and allowed to enter individualmicrowells. The excess sample solution remaining on the surface of themicrowell plate between openings into the microwells may be subsequentlyremoved from the microwell plate, which will restrict the presence ofthe sample to a vicinity of the individual reactive sites. Biochemicalreactions comprising dissociation of molecular complexes into individualsubunits or fragmentation of individual molecules into smallerfragments, e.g. proteolysis may benefit from the disclosed method.

In reference to FIG. 7E, a microarray reaction may be performed usingactive agents 745, which have been released from their correspondingcarrier microbeads 742 into a liquid medium and localized withinindividual spots on the microarray, such that the individual spots arefluidically disconnected from each other. In an embodiment, theindividual spot containing the released active agents comprises interiorof an individual microwell within a microwell array plate. In anembodiment, the step of releasing an active agent from its carriermicrobead comprises photolysis of a photosensitive linker between theactive agent and the microbead. Alternatively, the step of releasing anactive agent may comprise elution by acidic pH, by elevated temperature,by incubation with a digestive compound, time-dependent diffusion, etc.The step of releasing an active agent may occur either prior to orsubsequently to contacting the microarray 741 with a sample 744. In anembodiment, the active agents 745, which have been released from theircorresponding carrier bead into the liquid medium inside individualmicrowells, are capable of reacting with the sample 744 contacted withthe microarray. In this configuration, rather than performing a chemicalreaction using active agents conjugated to a microbead, a solution-phasechemical reaction may be performed in a microarray format.

In reference to FIG. 7F, a microarray reaction may comprise the steps ofsimultaneously contacting multiple beads with a sample prior to placingthe beads on a microwell plate, allowing the reaction to proceed for aspecific amount of time and subsequently arraying the reacted beads on amicrowell plate in spatially distinct locations for the subsequent beaddecoding and reaction readout. In this configuration, the reaction stepis spatially separated from the analysis step.

It should be understood that other methods of reacting a microarray witha sample may be implemented using the microarray compositions disclosedin the instant specification. One example is found in (Zhou, G., F.Khan, Q. Dai, J. E. Sylvester, and S. J. Kron. 2012. “Photocleavablepeptide-oligonucleotide conjugates for protein kinase assays byMALDI-TOF MS.” Mol Biosyst 8:2395-404), the entirety of which isincorporated herein by reference. The Zhou et al reference discloses aphotolabile peptide-oligonucleotide conjugate and microspheres bound toan oligonucleotide possessing a complementary sequence. Zhou et aldiscloses reacting the photolabile peptide-oligonucleotide constructwith a solution containing a protein kinase and subsequently capturingthe reacted (i.e. phosphorylated) construct on beads using thespecificity of the oligonucleotide binding to its complementarysequence. It further discloses subsequent peptide dissociation from thebeads by photoelution followed by analysis using MALDI MS. The type ofanalysis disclosed by Zhou et al may significantly benefit from theavailability of microarrays disclosed in the instant specification. Forexample, a multiplexed enzymatic assay may be designed that will includethe following steps: (i) providing several photolabilepeptide-oligonucleotide conjugates, (ii) providing multiple beadsindividually bound to oligonucleotide sequences, which are complementaryto the oligonucleotide sequences within the peptide-oligonucleotideconjugates, (iii) reacting the peptide-oligonucleotide conjugates withan enzyme-containing sample, (iv) capturing the reactedpeptide-oligonucleotide conjugates on beads using the annealing betweenthe complementary oligonucleotide sequences, (v) removing the unboundcomponents by washing the beads, (vi) making an array of beads on amicrowell plate, (vi) releasing the peptides from the beads byphotoelution and (vii) performing mass spectrometric analysis of thereleased peptides on the microwell plate.

Other types of multiplexed bead-based assays using the annealingreaction between the complementary oligonucleotide sequences may beimplemented using the disclosed microarray compositions. For example,oligonucleotide-conjugated beads may be used for purification andconcentration of DNA-tagged antibodies on beads.

Analysis of a Reacted Microarray

The U.S. patent application Ser. No. 13/369,939, Publication No.2012-0202709 A1, the entirety of which is incorporated herein byreference for the teachings therein, discloses multiple embodimentmethods of using optical spectroscopy and mass spectrometry to measureanalytes released from microbeads, which are positioned on a solidsupport, as well as analytes remaining on the microbeads. Many of theexperimental methods disclosed in the Ser. No. 13/369,939 applicationmay be utilized to measure the microarrays of the instant specification.In addition to MALDI TOF MS, various alternative methods of analytedesorption and ionization such as nanoparticle-assisted MS, liquid MALDIMS, matrix-free MS, API MS, DAPCI MS (desorption atmospheric pressurechemical ionization MS), LESA MS (liquid extraction surface analysisMS), LMJ-SSP (liquid-microjunction surface sampling probe), LSI (laserspray ionization), MAII (matrix assisted inlet ionization), DART (directanalysis in real time), SIMS (secondary ion mass spectrometry), LTP (lowtemperature plasma), ELDI (electrospray assisted laser desorptionionization), LAESI (laser ablation electrospray ionization), DESI(desorption electrospray ionization) and even the conventional ESI MS(electrospray ionization MS) may be used for measuring the microarraysof the present disclosure. Some of the above-references methods, e.g.LMJ-SSP are capable of quantitatively extracting the analyte from a spoton the microarray.

Depending on the specific method of mass spectrometry used to measurethe microarrays of the instant disclosure, the mass spectrometric dataacquisition may comprise acquiring mass spectra in either imaging ornon-imaging mode. Furthermore, the mass spectrometric data acquisitionmay be performed using the depth profiling capability of certain MSinstruments, such as LAESI and others. If such capability is utilized,it may be possible to generate a 3D (three-dimensional) image of anindividual microarray reactive site. A 3D image of a microarray reactivesite may provide valuable information related to the distribution of aparticular analyte both within the microwell and outside the microwellfollowing the analyte release from its carrier microbead.

Archiving and Storage of a Reacted Microarray

In an embodiment, the composite microarrays disclosed in the instantspecification allow analytes to be archived and stored on reactedmicroarrays for analysis performed at a later time point. In one aspect,this is possible because the amount of analyte present on the microarrayis greater than required for a single measurement. For example, thebinding capacity of a single 90 μm TENTAGEL® bead is approximately 1nmol of a peptide, while the amount of a peptide analyte sufficient fordetection by mass spectrometry is less than 1 pmol. Thus, a single spoton a microarray may contain as much as a 1,000-fold greater amount of ananalyte than needed for analysis.

It may however be advantageous to perform multiple measurements of thesame sample by mass spectrometry, for example to assess the sampleheterogeneity, detect the presence of single-site mutations,post-translational modifications, partially cleaved protein forms aswell as measure non-specific binding. Accordingly, it may beadvantageous to perform multiple measurements of the same microarrayspot by mass spectrometry using different instrument settings, e.g. thelinear, reflector and tandem MS mode, different mass range or evendifferent instruments, e.g. MALDI TOF and MALDI FT-MS. Such multiplemeasurements may be performed at different time points if the analytecan be preserved on the microarray. Furthermore, the microarrayarchiving may be required in the case involving clinical specimens.

The disclosed composite microarrays may be used for archiving analytesfor an extended period of time, e.g. several months or even severalyears. There disclosed two embodiment methods for archiving analytes ona microarray for subsequent analysis. In an embodiment, the analyte isreleased from a microarray reactive site, e.g. a microbead and localizedwithin a microarray analytical site (depicted as element 221 in FIG. 2),possibly mixed with MALDI ionization matrix. In an alternativeembodiment, the analyte is stored within a microarray reactive site,e.g. a microbead (depicted as element 222 in FIG. 2), so that only aportion of the analyte is initially released from the reactive site foranalysis. The U.S. patent application Ser. No. 13/369,939, PublicationNo. 2012-0202709 A1, the entirety of which is incorporated herein byreference for the teachings therein, discloses multiple sequential stepsof releasing analytes from a bead positioned on a microwell array plateand localizing the sequentially released analytes in the same spot onthe microwell array plate.

Microarrays suitable for the sample archiving and storage mayadditionally comprise a surface layer, which is chemically non-reactiveand does not adsorb analytes, for example a 10 nm layer of gold or othermaterial with similar relevant properties. The reacted microarrays maybe stored under conditions, which minimize the extent of chemicalreactions that lead to the analyte degradation. This may includepreventing effects such as photobleaching, oxidation, bacterialcontamination and others. Accordingly, the reacted microarrays may bestored at sufficiently low temperature, for example −20° C. or −80° C.,in a light-blocking container and under inert gas, e.g. nitrogen orargon.

Recovery of an Individual Microbead from a Microarray

In an embodiment, the composite microarrays disclosed in the instantspecification allow removing of one or multiple microbeads from theircorresponding positions within the microarray. The step of removing thebead from the microarray may occur after the step of releasing at leastsome of the analyte from the bead and analyzing the released analyte onthe microarray. For bead arrays fabricated using microwell array plates,the step of removing the bead from the microarray may compriseextracting the bead from its corresponding microwell. Individualmicrobeads, which have been removed from the microarray, may be furtheranalyzed outside the microwell plate or used in various downstreamapplications, e.g. DNA sequencing or PCR. Throughout this specification,the process of removing a bead from a microarray in a functional form,i.e. under conditions that make possible its subsequent use in adownstream application is referred to as a recovery of a bead from amicroarray.

In one aspect, the recovery of beads from the microarrays of the instantdisclosure is made possible by the disclosed microarray design. Asdescribed previously, the beads may be retained inside theircorresponding microwells simply by spatial constraints and withoutforming a covalent linkage with the solid support. The beads may be alsoretained inside individual microwells because of the substantial surfaceroughness of the sidewalls of the microwells. Alternatively, the beadsmay be retained inside individual microwells as a result of ahydrophobic interaction between the bead material and the inner surfaceof the microwells. The association between the bead and the microwell istherefore reversible and the beads may be removed from the microwells bymechanical or by other means, for example a solvent replacement. Inparticular, replacing a solvent with a solvent of different polarity mayaffect the bead swelling and accordingly the bead size. If the beads areretained inside the microwells by an applied magnetic field, removal ofthe magnetic field will allow facile separation of the beads from themicrowell plate.

The previously disclosed methods of analyte release from the beads,which are also disclosed in the U.S. patent application Ser. No.13/369,939, Publication No. 2012-0202709 A1, the entirety of which isincorporated herein by reference for the teachings therein, includephoto-release, release by acidic pH, release by a digestive compound,application of the MALDI matrix solution and competitive elution. Thesemethods are sufficiently mild to avoid causing substantial damage to thebead core or to the compounds conjugated to the bead. In the case ofdownstream MALDI MS analysis, the beads submerged into the microwellsare normally shielded from the high-intensity laser beam of the massspectrometer by a layer of the energy-absorbing MALDI matrix, whichlimits the radiation-induced damage to the beads. Therefore, largelyintact microbeads may be recovered from a microarray even after thesteps of releasing analytes from the beads and measuring the releasedanalytes by mass spectrometry. Alternative methods of analyte ionizationsuch as nanoparticle-assisted MS, liquid MALDI MS, matrix-free MS, APIMS, DAPCI MS (desorption atmospheric pressure chemical ionization MS),LESA MS (liquid extraction surface analysis), DART (direct analysis inreal time), LTP (low temperature plasma), ELDI (electrospray assistedlaser desorption ionization), LAESI (laser ablation electrosprayionization), DESI (desorption electrospray ionization) and evenconventional electrospray ionization (ESI MS), which utilizes acapillary for sample introduction into the mass spectrometer, are alsocompatible with the disclosed methods of recovery of beads from amicroarray. In fact, some of these methods utilize very mild conditions,e.g. ambient pressure and aqueous environment, which may help preservethe structure of beads made of agarose and other easily damagedmaterials.

It is possible that a layer of MALDI matrix, which is present on asurface of the microwell plate, may obscure positions of individualbeads or prevent their removal from the microwells. In such case, thematrix layer may be either depleted by the MS laser or simply washed offby rinsing the microwell plate surface with deionized water.

Certain additional procedures may be performed for recovering beads thatare fabricated from specific materials. For example, agarose beads,which have been exposed to the high vacuum of a MALDI TOF MS instrumentmay need to be rehydrated prior to their removal from the microwellplate, but such step may not be necessary for the polystyrene beads.

In an embodiment, beads that are sufficiently large, for example 100 μmdiameter or larger may be individually observed and manipulated using asuitable mechanical tool, e.g. a pipette tip, a needle, a Hamiltonsyringe, a magnetic picker or similar. Alternatively, the bead selectionand removal from a microarray may be performed using an automateddevice, for example a robotic spot picker, a bead picker, an automatedcolony picker available from Hudson Robotics Inc (Springfield N.J.) orsimilar. This process may be facilitated if a bead has distinctivefluorescence properties. It is also possible to remove only a fragmentof a bead, which nevertheless will contain enough analyte for thedownstream analytical applications.

The beads recovered from the bead microarray and compounds present onthe beads recovered from the bead microarray may be utilized in variousdownstream applications including, for example a Polymerase ChainReaction (PCR). PCR may be used, for example, to amplify DNA or RNAsequences from the beads used in the applications commonly known asin-vitro evolution and ribosome display. Alternatively, PCR or DNAsequencing may be used to elucidate the sequence of a bead-conjugatedaptamer that exhibit an affinity toward a particular biological target.In an embodiment, a peptide or a peptoid compound may be released fromthe bead recovered from a microarray for subsequent screening using asolution-phase chemical reaction.

In an embodiment, the disclosed composite microarrays enable recovery ofcertain compounds released from the individual beads positioned on amicrowell array plate for analysis performed outside the microwell platewhereas the corresponding carrier beads remain positioned on the plate.For example, DNA, RNA or other biomolecular analytes may be eluted froman individual bead or multiple beads onto the solid support in the formof a solution localized in a vicinity of the corresponding carrier bead.In this approach, certain surface properties of the solid support, e.g.greater hydrophobicity of areas located between openings into themicrowells may be useful to help localize the eluted analytes in thevicinity of their respective beads. The solution containing the analyteof interest may be subsequently removed from the solid support for aspecific downstream application, e.g. PCR.

Reactive Microbeads

There provided several non-limiting exemplary compositions of microbeadsthat may be utilized in the microarrays of the instant disclosure.Various modifications of the disclosed structures will be apparent to aperson skilled in the art.

In an embodiment, which is schematically shown in FIG. 8A, a microbeadmay comprise a solid support 801 and a capture agent 802 bound to thesolid support either directly or via an optional linker 803. The solidsupport 801 may be manufactured from any suitable material, e.g.agarose, polyethylene, polystyrene or a composite material and itsdimensions may vary. A non-limiting example of a suitable solid supportis a 90 micron diameter TENTAGEL™ bead. Multiple molecules of thecapture agent 802 may be bound to the solid support 801, for exampleapproximately 10¹² molecules, approximately 10¹⁴ molecules,approximately 10¹⁶ molecules, or other quantity. In an embodiment, thecapture agent 802 is a polypeptide, a protein or an antibody capable ofbinding a specific target, which is present or may be present in asample contacted with the microbead. In an embodiment, the capture agent802 is a monoclonal antibody. In an embodiment, the linker 803 is aprotein A or a protein G molecule. In an embodiment, at least 50% of thecapture agent molecules are bound to the solid support in the samemolecular orientation, for example a polypeptide capture agent may bebound to the bead via its C-terminal end. In an embodiment, at least 75%of the capture agent molecules are bound to the solid support in thesame molecular orientation. The capture agent 802 preferably has a knownchemical structure, e.g. an amino acid sequence, or at least a knowndigest profile. The digest profile of an analyte is an actual or insilico (theoretically calculated) mass spectrum acquired after theanalyte has been subjected to a reaction with a specific digestivecompound, e.g. trypsin. In the case of proteins, the digest profileusually comprises a series of signals, e.g. peaks in the low molecularweight region of the spectrum, e.g. below approximately 4,000 m/z. Aprotein digest profile is determined by the amino acid sequencecomposition of the precursor protein and therefore highly specific forindividual polypeptides and proteins. In an embodiment, the entirecapture agent 802 or at least a molecular portion thereof 804 is capableof reacting with a digestive compound contacted with the solid support801. For example, either a C-terminal or an N-terminal portion of apolypeptide, a protein or an antibody bound to a bead may be accessibleto a digestive compound contacted with the bead. In an embodiment, atleast 25% of a primary sequence of a polypeptide, a protein or anantibody, which is bound to the bead, may be accessible a digestivecompound, preferably at least 50% of a primary sequence, more preferablyat least 75% of a primary sequence, most preferably 100% of a primarysequence. In addition, the optional linker 803 may be accessible to andcapable of reacting with the digestive compound.

Microbeads with a structure similar to that depicted in FIG. 8A may beutilized in various bead-based bioassays without further means of thebead encoding, e.g. the beads may not require optical or mass tagencoding. In an embodiment, at least a portion of an amino acid sequenceof a proteinaceous capture agent 802 bound to the solid support 801 canbe measured by mass spectrometry and subsequently used to determine theidentity of the capture agent 802. In an embodiment, at least 25% of anamino acid sequence of a proteinaceous capture agent 802 bound to thesolid support 801 can be decoded by mass spectrometry, preferably atleast 50% of an amino acid sequence, more preferably at least 75% of anamino acid sequence, most preferably 100% of an amino acid sequence.

The measurement by mass spectrometry may comprise measurement of amolecular weight of the intact molecule, measurement of molecularweights of individual fragments generated by an enzymatic digest, i.e.peptide mass fingerprinting (PMF), measurement of molecular fragmentsgenerated by in-source decay (ISD), post-source decay (PSD) or by otherfragmentation mechanisms, MS-MS sequencing and other known methods. Inan embodiment, different capture agents within a library of microbeadsgenerate sufficiently different mass spectra after the reaction with adigestive compound. The mass spectra are subsequently analyzed todistinguish the different capture agents based on their characteristic“signature” series of peaks. This approach is illustrated in FIG. 8Cthat depicts an exemplary mass spectrum comprising several peaks 821,which correspond to the molecular weights of individual digestedfragments of the capture agent 802, depicted as m/z1, m/z2, m/z3, m/z4.A combination of three, four or a greater number of individual peaks821, which are measured with sufficiently high precision, e.g. withinapproximately 0.1 Da of the predicted position of a peak, may besufficient to distinguish different capture agents in the bead librariescomprising hundreds or even thousands of different compounds.

FIG. 8B schematically depicts a reacted microbead that is a microbead,which has been contacted with a sample. The reacted microbead depictedin FIG. 8B additionally comprises a target compound 811 bound to thecapture agent 802, for example a protein or a peptide antigen bound toits respective antibody. At least a portion 814 of the molecular complexcomprising the target compound 811 and the capture agent 802 bound tothe solid support 801 should be accessible to a digestive compound. Thefractional occupancy, i.e. the molar ratio of the target compound 811 tothe capture agent 802 may vary between individual beads. Depending on aconcentration of the target compound in the sample, all, some or none ofthe capture agents immobilized on a particular bead will be bound to thetarget 811. For example, approximately 50% of all capture agents 802bound to a particular bead may be also bound to the target 811.

FIG. 8D depicts an exemplary mass spectrum of a reacted microbead afterits exposure to a digestive compound. In addition to the peaks 821,which correspond to molecular fragments of the capture agent 802, aseries of new peaks 822 are also present in the mass spectrumcorresponding to molecular fragments of the target 811. The intensity ofpeaks corresponding to the capture agent 802 and to the target 811 inthe mass spectra acquired from the individual beads exposed to adigestive compound may be ratioed and subsequently used to measure thefractional occupancy of the binding sites 802 by the target 811.

An exemplary method of fabricating a reactive microbead conjugated to acapture agent (monoclonal antibody) with a known amino acid sequence isprovided below. 6% cross-linked agarose 34 micron diameter beadsfeaturing NETS-activated surface are available from GE Healthcare LifeSciences (Piscataway N.J.) as NETS HP SpinTrap. The beads are rinsed inseveral volumes of 1 mM HCl before reacting with an antibody. Anti-HSVmonoclonal antibody is available from EMD Biosciences (San DiegoCalif.). The antibody sequencing service is available from GenScript USA(Piscataway N.J.) or other commercial service providers. The antibody isdiluted to the 0.5 mg/mL concentration in a buffer containing 200 mMsodium bicarbonate and 200 mM NaCl. 10 μL of settled beads are combinedwith 20 μL of the anti-HSV antibody solution and incubated for 1 hourusing gentle rotation. Beads are washed with several volumes of a buffercontaining 200 mM sodium bicarbonate, 200 mM NaCl, 200 mM glycine and 1mM EDTA and then with several volumes of a pH 8 buffer containing 10 mMTris, 1 mM EDTA and 50 mM NaCl. The anti-HSV antibody conjugated beadsfabricated using the disclosed methods may be subsequently used for theisolation, concentration and purification of an HSV-tagged protein froma complex biological source.

Another non-limiting example of a microbead in which the identity of thebead-conjugated capture agent may be determined by mass spectrometryafter exposing the bead to a digestive compound is ANTI-FLAG® M2affinity gel available from Sigma-Aldrich (St. Loius Mo.). TheANTI-FLAG® M2 affinity gel comprises a purified monoclonal IgG1immunoglobulin, which is covalently bound to agarose microspheres by thehydrazide linkage. In this example, even though the primary amino acidsequence of the antibody may not be known to the end-user, a digestprofile of the bead-conjugated antibody may be easily obtained byreacting the affinity gel with an appropriate digestive compoundfollowed by the mass spectrometric analysis of the resulting proteindigest. It has been experimentally established that a sufficient numberof peaks, e.g. greater than three, which are specific to the antibody,are consistently detected in the mass spectra recorded from theANTI-FLAG® M2 beads, which have been in contact with 30 μg/mL aqueoussolution of trypsin for at least 15 min. Furthermore, mass spectra,which were recorded from the trypsin digest of different batches of theANTI-FLAG® M2 beads, have very similar spectral profile, i.e. theposition and relative intensity of the individual peaks are highlyreproducible in the mass spectra recorded from the different batches.

In an embodiment, which is schematically shown in FIG. 9A, a microbeadmay comprise: (1) a solid support 901, (2) a capture agent 902 bound tothe solid support either directly or via an optional linker and (3) areporter agent 903 bound to the solid support via a photolabile linker904. In an embodiment, the capture agent is an antibody, for example apolyclonal antibody or a monoclonal antibody. In an embodiment, thecapture agent is an anti-peptide antibody. In an embodiment, the captureagent is an aptamer, for example a DNA aptamer, an RNA aptamer or apeptide aptamer. The solid support 901 may be manufactured from anysuitable material, e.g. agarose, polystyrene or glass and its dimensionsmay vary. A non-limiting example of a suitable solid support is a 90 μmdiameter TENTAGEL™ bead. Multiple molecules of the capture agent 902 maybe bound to the solid support 901, for example 10¹² molecules, 10¹⁴molecules, 10¹⁶ molecules, or other quantity. Additionally, multiplemolecules of the reporter agent 903 may be bound to the solid support901, for example 10¹² molecules, 10¹⁴ molecules, 10¹⁶ molecules, orother quantity. In an embodiment, the capture agent 902 and the reporteragent 903 are bound to the solid support 901 in an approximatelyequimolar ratio. In an embodiment, the reporter agent 903 is apolypeptide, a peptidomimetic or a molecule with molecular weigh that isless than 2,000 Da.

The microbead compositions described in the previous paragraph may bereacted with a sample potentially containing a target, which in thisexample is a compound capable of binding to the bead-conjugated captureagent 902 with sufficiently high affinity. For example, the affinityconstant of a target binding to its respective capture agent may be inthe range between 10⁶M⁻¹ and 10⁹M⁻¹.

In reference to FIG. 9B, reacting the disclosed microbead compositionswith a sample containing a specific target may result in some, but notall of the capture agents binding and retaining the target analyte 905on the bead. In an embodiment, which is schematically shown in FIG. 9C,all or nearly all bead-conjugated capture agents within a particularbead contain the bound target analyte 905.

In an embodiment, the reporter agent 903 and the target analyte 905 havesimilar chemical structure. Non-limiting examples of compounds that havesimilar chemical structure are given below: (1) polypeptides that havean identical amino acid sequence but differ in the isotope compositionof individual amino acids, such as a 13C-enriched amino acid vs anatural isotope abundance amino acid vs a 13C-depleted amino acid; (2)polypeptides that have an identical amino acid sequence but additionallycontain a different group, such as a Cy3 or a Cy5 fluorescent label; (3)polypeptides that have closely related amino acid sequences, e.g. thosediffering in only a few positions within the polypeptide chain includingamino acid substitutions, additions and deletions; (4) polypeptides thathave an identical or very similar composition of amino acids, which arearranged in a different linear sequence; (5) polypeptides that have anidentical amino acid sequence but contain different post-translationalmodifications or alternatively contain identical post-translationalmodifications in different positions within an otherwise identical aminoacid sequence, for example a phosphorylated tyrosine. In an embodiment,the reporter agent 903 and the target analyte 905 have similarionization efficiency when analyzed by mass spectrometry.

Various methods that enable making of the microbead compositions of theinstant specification can be found in the art. As a non-limitingexample, the U.S. patent application Ser. No. 13/172,164, PublicationNo. 2012-0077688 A1, the entirety of which is incorporated herein byreference for the teachings therein, discloses several experimentalmethods that enable fabrication of microbeads conjugated to apolypeptide mass tag via a UV-photolabile linkage and additionallyconjugated to an anti-peptide monoclonal antibody (anti-HSV antibody)via covalent linkage. The Ser. No. 13/172,164 application does notdisclose that the polypeptide mass tags and the polypeptides captured bythe bead-conjugated monoclonal antibodies have similar chemicalstructure or similar ionization efficiency when analyzed by massspectrometry. Furthermore, the Ser. No. 13/172,164 application does notdisclose the possibility of quantitative measurement of the releasedanalytes by mass spectrometry.

The U.S. patent application Ser. No. 13/369,939, Publication No.2012-0202709 A1, the entirety of which is incorporated herein byreference for the teachings therein, discloses multiple embodimentmethods of either sequentially or concurrently releasing severaldistinct analytes from a single microbead positioned on a solid supportand localizing the released analytes within a single spot on the solidsupport, such that the transfer of the analytes from the bead onto thesolid support is performed quantitatively. The disclosed methods may beapplied for analysis of the microbead compositions described above.FIGS. 9D-9F schematically depict the mass spectra that may be recordedfrom the microbead compositions depicted in FIGS. 9A-9C, respectively.In particular, in the absence of binding of the target analyte to thebead, only the signal from the reporter agent 903 will be recorded, asdepicted in FIG. 9D. In the event of partial occupancy of the beadbinding sites by the target analyte, or alternatively their fulloccupancy, the mass spectrum will contain signals from both the targetanalyte and the reporter agent while the relative intensity of the twosignals will vary depending on the amount of the target analyteinitially bound to the bead.

The disclosed bead compositions may be particularly useful in variousassays aimed at measuring the concentration of specific peptidesgenerated by the proteolytic digest of complex biological mixtures, e.g.plasma, cell lysates and tissue lysates. Several mass spectrometry basedanalytical methods utilizing this approach are known in the artincluding methods termed SISCAPA™ and immunoMALDI or iMALDI™. The lattermethods can be implemented in the single-bead format, which is disclosedin the instant application.

Kinetic and time-dependent measurements performed on the beadmicroarrays analyzable by mass spectrometry

In an embodiment, the microarray compositions disclosed in the instantapplication enable repeated measurement of an analyte localized in aspecific position within the microarray using mass spectrometry. In anembodiment, the ability to perform repeated measurement of an analyte bythe sample-consuming technique, such as mass spectrometry, is madepossible because of a high analyte binding capacity of an individualmicrobead, for example 100 pmol per bead, 500 pmol per bead or 2 nmolper bead. The analyte binding capacity of a particular bead isdetermined by the bead material, the bead surface chemistry and the beaddimensions, among other parameters. For example, 300 μm diameterTENTAGEL™ beads have an approximately 2 nmol peptide binding capacity.Mass spectrometric analysis usually requires much lower amounts ofanalyte for detection, for example 100 amol, 1 fmol or 10 fmol,depending on the specific technique and the nature of the analyte.Therefore, the amount of analyte released from a single bead ispotentially much greater than the amount of analyte consumed during theanalysis by mass spectrometry.

As noted previously, the microarray compositions disclosed in thisapplication are inherently compatible with various types of biochemicalreactions including affinity binding, dissociation of a molecularcomplex, molecular diffusion, enzymatic digestion, modification of ananalyte chemical structure by an enzyme, e.g. kinase-mediatedphosphorylation of a peptide, etc. In an embodiment, the microarraycompositions disclosed in the instant application are compatible with:(1) biochemical reactions that occur on a solid support, e.g. on thesurface of a bead submerged into a microwell, and (2) biochemicalreactions that occur in a liquid phase, e.g. inside a liquid-filledmicrowell that contains an active agent released from a bead placed intothe microwell.

It is noted that many biochemical reactions may be performed in amicroarray format under conditions, which are inherently compatible withthe downstream analysis by mass spectrometry. That is, the compositionof the liquid medium including the concentrations of buffers, salts,detergents and other compounds will allow acquisition of high qualitymass spectra from the reacted microarrays, either during the course ofthe reaction or upon the reaction completion. In other words, the sampleclean-up procedures, such as desalting and detergent removal may beunnecessary in order to acquire good quality mass spectra from thereacted microarrays of the instant disclosure.

For example, an enzymatic proteolytic digestion reaction may beperformed in an aqueous medium, using 50 mg/mL concentration of anenzyme, such as trypsin or LysC dissolved in deionized water, or othersuitable medium e.g. 100 mM Tris buffer, PBS buffer, 100 mM ammoniumbicarbonate buffer, etc. Trace amounts of various other compounds, suchas urea, detergents, organic solvents, glycerol, etc may be also presentin the sample, as long as they are below a certain threshold.

For example, a kinase-mediated protein phosphorylation reaction mayinclude a peptide substrate, an enzyme (up to 50 nM), a co-factor (ATP,up to 10 μM), magnesium (mM range), buffers (HEPES or TRIS, up to 50mM), detergents (Tween-20 or CHAPS, up to 0.05%) and certain othercomponents. Such reaction compositions are inherently compatible withvarious methods of sample ionization for mass spectrometry, includingMALDI, DESI and LAESI and therefore may not need the sample clean-upprior to the MS analysis.

Certain methods of sample ionization for mass spectrometry, such asLAESI may be performed at ambient pressure, at relatively high humidity,e.g. between 5 and 95% humidity and also in a temperature range that iscompatible with occurrence of biochemical reactions, e.g. between 4° C.and 37° C. The liquid medium in which the reactions are performed, e.gwater is a suitable matrix for LAESI and several other sample ionizationmethods for mass spectrometry. The amount of water consumed during suchanalysis by mass spectrometry is relatively small, e.g. picoliters orless, which is much less than is present inside a single microwell.

In an embodiment, as schematically depicted in FIG. 10A, the disclosedmicroarray compositions enable multiple reactions to be performedsimultaneously on a same microarray. In an embodiment, individualreactions are largely localized within the space 1014 defined bydimensions of individual microwells. Such multiple reactions may beinitiated simultaneously, for example by simultaneously contactingmultiple reactive sites 1012 of a microarray 1010 with a sample.Alternatively, the multiple microarray reactions may be simultaneouslyinitiated by photolysis of a photosensitive linker between an activeagent and its corresponding carrier bead thereby releasing the activeagent into a liquid medium inside a microwell or by photolysis of aphotosensitive caged compound. Such simultaneously initiated reactionsmay then proceed for an extended period of time, e.g. 30 min, 1 hour, 6hours, 12 hours, 24 hours, etc before the reaction is terminated.

The analysis of a microarray by imaging or by non-imaging massspectrometry may be performed in a sequence of steps, in which theadjacent sample spots of the microarray are measured consecutively, bychanging position of the microarray solid support relative to theinstrument probing beam in specific pre-defined incremental steps, forexample in a snake-like pattern, as schematically illustrated in FIG.10B. The algorithms employed by the instrument data acquisition softwaremake it possible to repeatedly measure the same sample spot bypositioning the instrument probing beam at a pre-determined location,which matches the location of the sample spot, with sufficient precisione.g. within 1-10 microns of the actual spot location. Such positioningprecision is possible even if the microarray solid support has beentaken out of a mass spec instrument and subsequently placed back intothe instrument.

In an embodiment, as schematically depicted in FIG. 10C, multiplechemical reactions are simultaneously initiated on a microarray butsubsequently terminated or stopped at different time points. In anembodiment, the reaction termination is achieved by contacting anindividual microwell or a group of microwells with an acidic compound,e.g. 1% solution of trifluoroacetic acid (TFA) or similar. This may beperformed using a liquid dispensing instrument or by manual pipetting.The reactions, which have been initiated at the same time but terminatedat different time points, may be subsequently measured by massspectrometry directly on the microwell plate, thereby enabling analysisof a time course of a specific biochemical reaction.

In an embodiment, as schematically depicted in FIG. 10D, it is possibleto acquire mass spectrometric data from a microarray of the instantdisclosure during the course of a chemical reaction, which occurs on themicroarray, without having to terminate such chemical reaction prior toacquisition of the mass spectrometric data from the microarray. In otherwords, it is possible to sample a chemical reaction in real time, as itoccurs on a microarray, by mass spectrometry. As schematically depictedin FIG. 10D, the acquisition of the mass spectrometric data may beperformed at specific, pre-determined time points such that a singlesample spot is measured multiple times, e.g. at time points T1, T1′,T1″, etc. Because the mass spectrometric data acquisition process isusually fast, e.g. an averaged spectrum is acquired in less than 1second, it is possible to measure hundreds or even thousands ofindividual spots on a microarray within 1 hour or less and subsequentlyrepeat data acquisition from the previously measured spots.

As a non-limiting example of how such time-dependent data acquisitionprocess may be implemented, the instant specification discloses severaltypes of microwell array plates suitable for performing massspectrometry analysis including microwell plates suitable for analysisby Desorption Electrospray Ionization (DESI), Laser AblationElectrospray Ionization (LAESI), Liquid Microjunction Surface SamplingProbe (LMJ-SSP) and/or Liquid Extraction Surface Analysis (LESA) massspectrometry. The above-referenced mass spec techniques are capable ofanalyzing liquid as well as solid samples and can work with a variety ofsolid supports including solid supports made of glass, polymers andcomposite materials. The volume of a liquid medium confined withinindividual microwells on a microwell plate may range from less than 1microliter to 1 milliliter or greater, for example when utilizing theindustry-standard 96-, 384- and 1536-well microwell plates. Individualwells of a microwell plate may include one or more microbeads, eachmicrobead containing from approximately 1 pmol to approximately 1 nmolor more of an active agent, e.g. a polypeptide or a protein. The activeagent may be released from the beads into the liquid-filled microwellsusing UV photoelution or by other methods, optionally followed by mixingthe released active agent with the liquid medium inside the microwell.The liquid medium may contain a reactive moiety capable of reacting withthe active agent, e.g. a digestive enzyme such as trypsin. The activeagent released from the bead into the liquid-filled microwell may berepeatedly measured by mass spectrometry, either in its original(unmodified) form or during the course of a chemical reaction, e.g.enzymatic digestion. Furthermore, the established methods of mass specanalysis such as LAESI, are capable of performing sample depthprofiling, which may be particularly useful for identifying 3D (threedimensional) distribution of an analyte following its release from thecarrier bead.

In an embodiment, the microarrays of the instant disclosure areconfigured for performing kinetic analysis of chemical reactions, whichoccur on the microarray, using optical spectroscopy, e.g. fluorescent,luminescent or colorimetric spectroscopy. The optical analysis may beperformed using a microarray scanner, a fluorescence microscope or othersimilarly functioning device, e.g. a Roche 454® DNA pyrosequencinginstrument. In particular, many models of fluorescence microscopes, e.g.NIKON® inverted TE2000 microscope may be equipped with a temperature,CO₂ and humidity-controlled environmental chamber, which enablesperforming a chemical reaction on a microarray under specificenvironmental conditions while simultaneously acquiring optical datafrom the microarray. In an embodiment, the chemical reactions occur in athree-dimensional space, e.g. inside a liquid-filled microwell.Accordingly, it may be advantageous to utilize optical imaginginstruments featuring adjustable focus distance to generate a series ofthree-dimensional images of a reactive microarray, which may be taken atspecific time points. A non-limiting example of such instrument isNIKON® inverted TE2000 microscope equipped with prior motorized X, Ystage and the focus motor to capture Z-stacks of the microarray. Anon-limiting example of a camera capable of capturing microarray imagesis HAMAMATSU® ORCA ER digital CCD camera. A non-limiting example of adata acquisition software capable of acquiring three-dimensionalmicroarray images is NIKON® Elements computer software.

FIGS. 11A-11B provide further illustration into how bead microarrayreactions may be terminated at different time points and subsequentlyanalyzed by mass spectrometry and/or optical spectroscopy directly on asolid support, which is used for the microarray fabrication. Asdisclosed previously, e.g. in FIG. 1, the microwell plates used for thefabrication of reactive bead microarrays may be subdivided into “pads”or sub-arrays of microwells. For example, some experiments disclosed inthe instant specification utilized microwell plates that contained 16(8×2) sub-arrays, each sub-array containing 169 (13×13) microwellswithin a 7×7 mm area. In an embodiment, the microwell plate issubdivided into the sub-arrays of microwells by affixing a gasket to thetop surface of the microwell plate. A suitable gasket is a 12-wellself-adhesive silicone gasket available from Ibidi LLC (Verona Wis.) asa part of the cell culture chamber. Each of the 12 wells of the Ibidigasket measures approximately 7.5 mm×7.5 mm×8 mm and holds approximately250 μL of a liquid medium. As schematically illustrated in FIG. 11A,affixing the gasket to the top surface of a microwell plate, whichcontains an array of reactive microbeads, will create multiplemicroarray compartments. As schematically illustrated in FIG. 11B, someor all of the microarray compartments may be subsequently filled with asample-containing liquid medium. The experimental reaction conditions,e.g. the composition of the liquid medium may be identical or differbetween the different microarray compartments. The gasket attached tothe surface of a microwell plate may form a watertight seal.Accordingly, as schematically illustrated in FIG. 11C, the liquid mediummay be subsequently removed from the individual microarray compartments,optionally followed by rinsing with deionized water, thus effectivelyterminating the microarray reaction inside a specific microarraycompartment while allowing the reaction to proceed inside othermicroarray compartments. The ability to terminate microarray reactionsat different time points may be used for studying reaction kinetics.

Two- and Three-Dimensional Imaging of a Microarray

Some of the microarrays disclosed in the instant specification may bedescribed as three-dimensional (3D) microarrays, which have thefollowing characteristics: (i) the microwell plates are utilized asthree-dimensional solid supports; (ii) the active agent-conjugatedmicrobeads function as three-dimensional reactive sites and (iii) theanalytes released from the reactive sites are localized inthree-dimensional regions for the downstream analysis by one or severalanalytical methods. Accordingly, in some cases it may be beneficial toapply methods of 3D optical imaging to characterize such arrays, eitherin addition to or instead of the more conventional two-dimensional (2D)imaging methods. For example 3D optical imaging may be utilized to probethe position of individual beads within individual microwells or toprobe the localization of analytes, which have been released fromindividual beads. The 3D optical imaging of a microarray may beperformed before or after reacting the microarray with a sample. In anembodiment, the optical imaging is fluorescence imaging. In anembodiment, the 3D fluorescence imaging of a microarray is performedusing a microarray scanner or a fluorescence microscope. Severalnon-limiting examples of 3D imaging of a microarray are provided in theExperimental Examples section of the specification.

In an embodiment, the microarrays disclosed in the instant specificationare analyzable using the methods of mass spectrometry imaging (MSI), forexample MALDI TOF MSI. The MS imaging of a microarray may be performedeither in addition to or instead of the conventional non-imaginganalysis by mass spectrometry, in which analyte-containing spots areindividually measured by mass spectrometry. Several non-limitingexamples of mass spectrometric imaging of a microarray are provided inthe Experimental Examples section of the specification.

Furthermore, various methods of 3D mass spectrometry may be applied tomeasure the microarrays of the instant disclosure. A non-limitingexample of a 3D mass spectrometry is 2D lateral mass spectrometricimaging of a sample combined with the sample depth profiling, which maybe performed at ambient pressure using the LAESI technology,specifically on the LAESI® DP-100 instrument. The 3D mass spectrometricimaging of a microarray may be performed in order to measure spatialdistribution of an analyte after its release from a carrier bead.

Overall, various options available for analyzing the microarrays of theinstant disclosure are depicted in FIG. 12. The double-sided arrowssignify the fact that the 2D and 3D analysis, as well as the optical andmass spectrometric analysis may be performed in either order and thatthe analysis of both an unreacted and reacted microarray, i.e. prior toand after the microarray contact with a sample is possible.

Analysis of Bead-Conjugated Analytes Comprising a Nucleic Acid

In an embodiment, the microarrays disclosed in the instant specificationare suitable for determining the sequence of a nucleic acid, e.g. RNA orDNA, which are bound to the beads either directly or through a linker,which may be also an analyte. FIG. 13 schematically illustrates severaloptions available for decoding the sequence of a nucleic acid. Asdisclosed elsewhere in this specification, an array 1301 comprisinganalyte-conjugated beads positioned on a microwell plate can beconverted into a combination of a bead array and an array of microspots1302 by eluting analytes from the beads and localizing the elutedanalytes in discrete spots in the vicinity of their corresponding beadson the microwell plate. The released analytes are independentlyanalyzable by mass spectrometry and optical spectroscopy while the beadsfrom which the analytes have been eluted are analyzable by opticalspectroscopy.

The nucleic acid analytes may be either eluted from the beads or remainbound to the beads. In the former case, mass spectrometry may be used toperform nucleic acid sequencing using various experimental proceduresknown in the art. It is also possible to withdraw an aliquot containingthe eluted nucleic acid analyte from the microwell plate for PCRanalysis. These options are indicated using the UP arrow 1311. However,it is also possible to sequence the nucleic acid analytes, which remainbound to the bead, as indicated by the DOWN arrow 1312. In particular,the bead arrays disclosed in the instant specification enable selectiveremoval of one or multiple beads from the microwell plate for thepurpose of further analysis, which may include PCR or nucleic acidsequencing including various techniques of next-generation DNAsequencing. The specific beads to be removed from the microwell platemay be selected based on their optical, e.g. fluorescence properties oralternatively on the basis of the mass spectrometric data or opticaldata acquired from the eluted analytes. Yet another option is to performDNA analysis directly on the beads positioned on the microwell plate. Inan embodiment, both the beads (e.g. 34 micron agarose beads) andmicrowell plates (e.g. fiber optic plates featuring 42 micron diametermicrowells) are similar to those utilized on the Roche 454® DNApyrosequencing platform. The beads containing the nucleic acid sequencesmay remain essentially intact after analysis by mass spectrometry whensufficiently mild, ambient pressure mass spec techniques are utilizedsuch as LAESI or DESI.

Fabrication and Analysis of Arrays Comprising Paramagnetic orFerromagnetic Beads

In an embodiment, the instant specification discloses microarraycompositions and experimental procedures that include magnetic beads.Magnetic beads are widely used in biomedical research including variousgenomic and proteomic applications and in sample analysis by massspectrometry. Several novel aspects are disclosed below. In anembodiment, the magnetic beads are paramagnetic, super paramagnetic orferromagnetic. In an embodiment, the magnetic beads are sufficientlylarge to enable single bead analysis by mass spectrometry, for examplethe beads may be capable of binding and subsequently releasing at least10 pmol of a peptide analyte for analysis by mass spectrometry, morepreferably at least 100 pmol of a peptide analyte. Such high-capacitybeads may be approximately 10 μm diameter or greater, preferablyapproximately 20 μm diameter or greater, more preferably approximately50 μm diameter or greater, most preferably approximately 100 μm diameteror greater. In an embodiment, the magnetic beads are either monodisperseor have sufficiently narrow size distribution to allow positioning ofone bead per microwell on a microwell array plate. In an embodiment, themagnetic beads have suitable surface chemistry, which allows conjugationof biomolecules to the bead surface. In an embodiment, the magneticbeads have surface chemistry, which allows performing on-bead chemicalreactions, such as affinity binding or an enzymatic reaction. The latterrequirement may be satisfied by the presence of an appropriate spacerand/or linker such as poly-glycine or a PEG molecule. In an embodiment,the magnetic beads have fluorescent properties. Methods of making suchbeads are known in the art. Beads matching the above requirements arealso available commercially. For example, Spherotech, Inc (Lake Forest,Ill.) offers various ferromagnetic and paramagnetic beads with respectto the bead diameter, surface chemistry and fluorescence properties.Detailed description of the magnetic beads is available online on theSpherotech website (accessed Apr. 24, 2013) and off-line from theirproduct catalog. For example, Spherotech catalog number FCM-100052-2beads are fluorescent yellow carboxyl magnetic particles with 90-105 μmnominal size. The FCM-100052-2 beads can be used for making a bead arraywith one bead per well occupancy on a microwell plate with cylindricalmicrowells approximately 120 μm wide and 120 μm deep. Spherotech catalognumber SVMH-500-4 beads are streptavidin coated magnetic particles with45-52 μm nominal size. Spherotech catalog number CFM-1000-5 beads arecarboxyl ferromagnetic particles with 90-120 μm nominal size.

An exemplary experimental workflow that allows single bead analysis bymass spectrometry and/or fluorescence in a bead array format isdisclosed below. The individual beads may be conjugated to activeagents, such as peptides, antibodies, aptamers, proteins,oligonucleotides, etc and may have unique optical properties, e.g.fluorescence emission spectra. The individual beads may be a part of acombinatorial bead library. Multiple reactive beads combined in a singlereaction volume form a multiplexed suspension bead assay. In referenceto FIG. 14A, it is noted that a multiplex reaction comprising multiplemagnetic beads may be performed in a single reaction vessel, e.g. anEPPENDORF™ microcentrifuge tube and upon the reaction completion thelarge magnetic beads may be subsequently washed and rinsed usingprocedures commonly utilized for smaller, e.g. micron-size magneticbeads. For example, the beads 1401 may be collected on a side of themicrocentrifuge tube 1402 using a conventional magnetic separation rack1403, available for example from GenScript (Piscataway, N.J.) as catalognumber M00112. In reference to FIG. 14B, it is noted that the reactedand rinsed beads 1411 may be subsequently transferred onto a microwellarray plate 1412 and distributed into individual microwells 1413 byplacing a suitable magnet 1414 underneath the bottom surface of themicrowell plate and moving the magnet across the microwell plate to dragthe beads along. The size-matching microwells accept one bead per well1415 and the magnet may continue to be moved until the majority or allthe beads are distributed into the microwells. The excess beads that didnot go into the microwells may be subsequently removed from the plate.The previously disclosed methods of eluting analytes from the beadsfollowed by analysis of the eluted analytes and/or analysis of the beadsby a spectrographic method may be utilized with the bead arrayscomprising magnetic beads.

In reference to FIG. 14C, it is noted that the disclosed magnetic beadarray compositions may enable selective removal of one or several beadsfrom a selected location within a microwell plate for a downstreamanalysis, e.g. by PCR while retaining the remaining beads on themicrowell plate. This may be accomplished by: (i) identifying locationof a bead with specific properties on a microwell plate. The specificproperties may include specific fluorescence or mass spectrometricproperties; (ii) positioning a magnetic bead picker or a similarlyfunctioning device 1421 near the surface of a microwell plate andsufficiently close to the location identified in the previous step. Forexample the magnetic bead picker may be positioned on the (X, Y) planewithin 50 μm, within 250 μm or within 1 mm from the identified location;(iii) bringing the magnetic bead picker or a similarly functioningdevice 1421 sufficiently close on the Z axis (height) to a bead presentin the identified location such that only one bead or a few beads 1422attach to the picker; (iv) transferring the picked bead(s) to a separatelocation. A suitable magnetic bead picker is PickPen II 1-M MagneticDevice available from Sunrise Science Products, Inc (San Diego, Calif.)as the catalog item 73090.

It is noted that the disclosed compositions and experimental proceduresutilizing magnetic beads may bring about one or more of the followingbenefits: (i) eliminate the need for centrifugation because the beadhandling and separation is achieved by a magnet; (ii) provide enhancedpositional stability for bead arrays on a microwell plate, particularlywhen the beads are only loosely bound to the surface of the microwellplate. In other words, the magnetic beads will tether to the plate dueto the presence of a magnetic field. This may be particularly importantfor the microwell plates featuring microwells with a lower depth/widthaspect ratio (“shallow wells”); (iii) provide facile automation andintegration of multiple analytical steps, e.g. reacting beads with asample, bead wash, bead rinse, bead array fabrication, bead picking on asingle instrument platform.

It is noted that while MALDI MS is a perfectly suitable method of massspectrometric analysis of bead arrays, alternative techniques such asLAESI and DESI, which feature ambient pressure ionization, compatibilitywith aqueous chemistry and a more gentle analyte desorption mechanism,which is compatible with various surface materials, may offer uniqueadvantages particularly when the bead recovery from a bead array isdesired.

Living Cell Microarrays

The following sections describe devices, methods and compositionsrelated to the fields of biological cell culture, high-throughput cellscreening, cell microarrays, bead-based cell assays, live cellscreening, intact cell analysis, fluorescence microscopy and whole cellmass spectrometry.

Various devices suitable for cell culture are known in the art. Examplesof the commonly utilized devices are a Petri dish and culture multi-wellplates, such as 6-well, 12-well and 24-well plates. The plates areusually made of biocompatible plastic or glass. The single- andmulti-well plates enable cultivation of anchorage-dependent (adherent)cells, as well as suspension cell cultures and can be utilized innumerous downstream applications. The adherent cell culture andsuspension cell culture assays can be performed either in a manual or anautomated fashion.

Devices are known in the art that integrate cell culture assays with thedownstream analysis of the cultured cells by optical methods, forexample microscopy of in situ cell culture and immunofluorescencestaining. Such devices are sometimes referred to as chamber slides orcell chamber slides and usually comprise a non-fluorescent solidsupport, for example a glass microscope slide, a removable multi-wellchamber or a multi-well gasket affixed to the solid support and a lid.The multi-well chamber is made of biologically inert plastic, silicon orother material with similar relevant properties. The multi-well chambersubdivides the slide into multiple regions or assay locations so thatdifferent cell culture conditions, for example different concentrationsof a bioactive compound may be simultaneously tested on the slide. Theadherent cells growing on the surface of the slide can be measureddirectly on the slide. Examples of the commercially available cellchamber slides are Millicell EZ slides from Millipore (Billerica Mass.)and Lab-Tek chamber slides from Thermo Fisher (Waltham Mass.).

Further miniaturization of living cell assays is achieved by the cellmicroarray technology, which can accommodate thousands of reactive siteson a single 25×75 mm microchip. In one approach, a large number ofdifferent compounds such as proteins, antibodies, lipids or DNA areimmobilized, e.g. printed on a flat surface of a glass microchip andincubated with a cell suspension. Cells with affinity to a particularcompound are retained in individual spots on the microchip. In adifferent approach, which is sometimes referred to as a reversetransfection microarray or a microarray gene expression system,eukaryotic cells are cultured on a microchip that features multiplespots containing different DNA sequences cloned into a mammalianexpression vector. The cells are transfected directly on the microchipand the DNA uptake and expression is monitored by optical analysis ofthe cell clusters on the microchip. A description of this method, whichhas been successfully used for screening cDNA and RNAi libraries, may befound for example in (Ziauddin, J., and D. M. Sabatini. 2001.“Microarrays of cells expressing defined cDNAs.” Nature 411:107-10).

Devices and methods that enable in situ analysis of cell arrays by oneor several analytical methods are advantageous because they provide morestreamlined approach to the high-throughput cell screening andfacilitate assay automation. Accordingly, the instant specificationdescribes various devices, compositions and methods for culture and insitu analysis of biological cells by analytical methods, which mayinclude optical and mass spectrometric analysis, mass spectrometricimaging and whole cell mass spectrometry. The disclosed devices,compositions and methods may be utilized in a wide range of cell-basedassays, non-limiting examples of which are RNAi transfection, proteintranslocation, micronuclei assays, cytotoxicity, cell activation, celldifferentiation, ADME and toxicity, GPCR signaling and cell-cellinteraction.

In an embodiment, the disclosed compositions comprise a multi-wellchamber reversibly affixed to a surface of a solid support, wherein thesolid support is configured for analysis of biological cells by massspectrometry. In an embodiment, the solid support is a microscope slideor a microwell array plate. In an embodiment, the solid support isconfigured for analysis of intact cells by both mass spectrometry andoptical microscopy.

In an embodiment, the disclosed compositions comprise a multi-wellchamber reversibly affixed to a microwell array plate, in which thesurface between openings into the microwells, the sidewalls ofindividual microwells, the bottom of individual microwells, or anycombination of the above are configured for attachment of adherentcells.

In an embodiment, the disclosed compositions comprise a microwell arrayplate and a plurality of microbeads distributed into individualmicrowells of the microwell array plate. In an embodiment, themicrobeads are individually conjugated to one or more distinct activeagents and the active agents may be releasable from the beads arrayed onthe microwell plate. In an embodiment, the disclosed compositions areconfigured for cell culture and subsequent analysis of the culturedcells by optical microscopy, mass spectrometry or both.

In an embodiment, the disclosed compositions comprise a plurality ofmicrobeads configured for binding a specific number of biological cellsand a microwell array plate configured for accepting the microbeads withthe biological cells bound thereto into individual microwells. In anembodiment, the surface of the microbeads is configured for bindingadherent cells. In an embodiment, the surface of the microbeadscomprises an affinity agent capable of binding a specific population ofadherent or non-adherent cells. The affinity agent may be a protein,such as an antibody, a peptide, a nucleic acid, a lipid, a carbohydrateor another biomolecule. An individual microbead may also comprise anoptical, e.g. fluorescent label or a mass tag, which may be subsequentlyused to determine identity of the affinity agent present on themicrobead. In an embodiment, the active agent or their fragments arereleasable from the beads and analyzable by mass spectrometry.

In an embodiment, the disclosed composition is a cell concentrationdevice comprising a multi-well chamber reversibly affixed to a solidsupport, which may be a microscope slide or a microwell array plate. Thedisclosed device is configured for accumulating biological cells, whichare initially present in a cell suspension, on or near the surface ofthe solid support for the downstream analysis by mass spectrometry. Inan embodiment, the accumulation of cells on the solid support isachieved using centrifugation.

Application of Whole Cell Mass Spectrometry to the Analysis ofBiological Cells

Whole cell mass spectrometry (WCMS), which is also known as intact cellmass spectrometry (ICMS), is an analytical method that enablesmeasurement of contents of biological cells without prior fractionationof individual analytes or using minimal analyte fractionation. Methodsof WCMS that utilize the Matrix-Assisted Laser Desorption-Ionizationmechanism are known as “whole cell MALDI mass spectrometry” or “wholecell MALDI”. Other methods of analyte ionization for the whole cell massspectrometry are also known including, for example, nanostructureinitiator MS, DESI MS, nano-DESI MS and LAESI MS. In general, both laserdesorption ionization MS and electrospray ionization MS, as well asvarious modifications of these techniques are suitable for analyzingbiological cells.

The majority of biological cells are amenable to the WCMS analysis,including bacterial cells, eukaryotic cells, mammalian cells, humancells, organ- and tissue-specific cells, disease-specific cells,pathway-specific cells, drug-treated cells, plant cells, fungal sporesand other types of cells. The cells may be cultured in a laboratory;alternatively the cells may be isolated from an environmental source,such as air, soil, seawater or fresh water. Furthermore, the cells mayoriginate from a clinical specimen.

In a typical WCMS protocol, a sample for analysis is prepared by mixingintact cells, which are suspended in water, ethanol, buffer solution orother suitable medium, with a solution containing dissolved MALDImatrix, such as CHCA, SA or DHB. The mixing may be performed in amicrovial or directly on the MALDI target plate. Several hundred or evenseveral thousand individual cells may be used to prepare one sample,although as little as one cell may also generate a signal detectable bymass spectrometry (Boggio, K. J., E. Obasuyi, K. Sugino, S. B. Nelson,N. Y. Agar, and J. N. Agar. 2011. “Recent advances in single-cell MALDImass spectrometry imaging and potential clinical impact.” Expert RevProteomics 8:591-604). The MALDI matrix solution usually contains anacid and/or organic solvent, such as acetonitrile or methanol, which maycause rupture of the cell membrane and partial release of the cellularcontents. Upon evaporation of the solvent, the MALDI matrixcrystallizes, the cells and their contents become embedded into thematrix crystals and are ready for the mass spec analysis.

Numerous applications of WCMS are known, in particular in the areas ofenvironmental monitoring, clinical diagnostics and metabolic profiling.

U.S. Pat. No. 6,177,266 discloses a method of identifying bacteria, i.e.identifying the genus, species and strain using biomarker-specific peaksobserved in the mass spectra acquired from cellular protein extracts orfrom whole cells.

U.S. Pat. No. 7,865,312 discloses identification of metabolites in thewhole cell samples using the method of FT-MS.

Hazen et al (Hazen, T. H., R. J. Martinez, Y. Chen, P. C. Lafon, N. M.Garrett, M. B. Parsons, C. A. Bopp, M. C. Sullards, and P. A. Sobecky.2009. “Rapid identification of Vibrio parahaemolyticus by whole-cellmatrix-assisted laser desorption ionization-time of flight massspectrometry.” Appl Environ Microbiol 75:6745-56) discloses applicationof MALDI TOF MS to identify a pathogenic strain of the marine bacteriumVibrio parahaemolyticus and distinguish it from the closely relatedbacterial strains using the method of WCMS.

Gagnaire et al (Gagnaire, J., O. Dauwalder, S. Boisset, D. Khau, A. M.Freydiere, F. Ader, M. Bes, G. Lina, A. Tristan, M. E. Reverdy, A.Marchand, T. Geissmann, Y. Benito, G. Durand, J. P. Charrier, J.Etienne, M. Welker, A. Van Belkum, and F. Vandenesch. 2012. “Detectionof Staphylococcus aureus Delta-Toxin Production by Whole-Cell MALDI-TOFMass Spectrometry.” PLoS One 7:e40660) discloses application of WCMS todetect the Staphylococcus aureus delta-toxin in intact bacterial cellsand further, to correlate expression of the delta-toxin with theaccessory gene regulator status by using isogenic wild-type and mutantstrains of the bacterium.

Kulkarni et al (Kulkarni, M. J., V. P. Vinod, P. K. Umasankar, M. S.Patole, and M. Rao. 2006. “Intact cell matrix-assisted laserdesorption/ionization mass spectrometry as a tool to screen drugs invivo for regulation of protein expression.” Rapid Commun Mass Spectrom20:2769-72) discloses application of WCMS to detect expression of arecombinant protein in the E. coli cells and suggests that WCMS can beused to screen for drugs, which regulate protein expression, as well asfor drugs that affect protein localization and protein conformation.

Dong et al (Dong, H., W. Shen, M. T. Cheung, Y. Liang, H. Y. Cheung, G.Allmaier, O. Kin-Chung Au, and Y. W. Lam. 2011. “Rapid detection ofapoptosis in mammalian cells by using intact cell MALDI massspectrometry.” Analyst 136:5181-9) discloses application of WCMS todetect specific differences in the mass spectra obtained from theliving, necrotic and apoptotic mammalian cells. The apoptosis-specificpeaks were similar between the different cell lines and also similarbetween cells that have been exposed to different apoptosis-causingdrugs. Furthermore, it was shown that intensity of theapoptosis-specific mass peaks as measured by WCMS increasesproportionally to the fraction of the apoptotic cells in the cellpopulation.

Hanrieder et al (Hanrieder, J., G. Wicher, J. Bergquist, M. Andersson,and A. Fex-Svenningsen. 2011. “MALDI mass spectrometry based molecularphenotyping of CNS glial cells for prediction in mammalian braintissue.” Anal Bioanal Chem 401:135-47) discloses correlation betweenpeaks in the mass spectra recorded from the mammalian brain tissueimaged by MALDI TOF MS and the whole cell mass spectra recorded fromcultured neural cells.

Molecular weight of compounds that can be detected by WCMS varies fromless than 100 Da to several hundred kDa or greater. In practice, anarrower MW range is usually measured in a single experiment. Smallmolecules with MW of several hundred Da, such as metabolites, drugcompounds, lipids, etc may be measured in the MALDI TOF reflector mode.Larger molecules, such as polypeptides and intact proteins may bemeasured in the MALDI TOF linear mode. At present, the majority ofpolypeptides and proteins that are reliably detected by MALDI TOF WCMShave molecular weight that is less than approximately 20 kDa, althoughthere are some exceptions. Signals from hundreds or even thousands ofdistinct analytes may be detected in a single mass spectrum. The analytedetection may be performed in either positive or negative ion mode.

There is a marked difference between the ability of WCMS to detect lowmolecular weight compounds, such as metabolites and lipids and highermolecular weight compounds, such as proteins. The observed differencemay be related to the greater ionization efficiency of the low MWcompounds compared to the higher MW compounds. It may be also related tothe fact that the low MW compounds, such as metabolites are present in acell in a much higher molar concentration (several orders of magnitudehigher) compared to even very abundant proteins, for example ubiquitin.Consequently, the detection of polypeptides and proteins requiressignificantly greater amount of starting material compared to thedetection of small molecules. For example, WCMS can detect and evenquantify small molecule metabolites, e.g. ADP, ATP and GTP, which areextracted from a single cell; in contrast WCMS of polypeptide andprotein analytes performed in the 2,000-20,000 Da MW range may require asample comprising more than 100 individual cells in order to obtain ahigh quality mass spectrum (Dong, H., W. Shen, M. T. Cheung, Y. Liang,H. Y. Cheung, G. Allmaier, O. Kin-Chung Au, and Y. W. Lam. 2011. “Rapiddetection of apoptosis in mammalian cells by using intact cell MALDImass spectrometry.” Analyst 136:5181-9).

MALDI TOF mass spectra of a few highly abundant metabolites and lipidscan be acquired from single cells positioned on an electricallyconductive surface (Urban, P. L., K. Jefimovs, A. Amantonico, S. R.Fagerer, T. Schmid, S. Madler, J. Puigmarti-Luis, N. Goedecke, and R.Zenobi. 2010. “High-density micro-arrays for mass spectrometry.” LabChip 10:3206-9). In this approach, a diluted suspension of biologicalcells is distributed among multiple hydrophobic spots by repeatedpipetting and the areas containing single cells are manually selectedfor MS analysis.

Microarrays are known in the art that enable analysis of individual,i.e. single cells by optical imaging or by other analytical methods. Thedevices, methods and compositions disclosed in the instant specificationenable analysis of single cells in a microarray format by WCMS, eitheralone or in combination with other analytical methods, e.g. opticalimaging. Furthermore, the devices, methods and compositions disclosed inthe instant specification enable analysis of cell populations comprisingmultiple cells in a microarray format by WCMS, either alone or incombination with other analytical methods. Furthermore, the devices,methods and compositions disclosed in the instant specification enableanalysis of cell populations, which comprise a specific number ofindividual cells, e.g. approximately 10 cells, approximately 100 cells,approximately 1,000 cells or another number. Currently, the single-cellanalysis by WCMS is utilized primarily for detection of the mostabundant cellular components, such as ATP, other common metabolites andcertain lipids. In contrast, the devices, methods and compositionsdisclosed in the instant specification have the ability to substantiallyincrease the sensitivity of single cell WCMS and to enable detection ofa greater number of analytes including certain polypeptides, proteinsand other compounds in a single cell. Up to now, detection ofpolypeptides, proteins and related compounds by WCMS in a single cellhas been either very difficult or impossible, in part due to theintrinsically low concentration of these analytes within the cell. Theability to detect or quantify specific peptides or proteins using singlecell whole cell mass spectrometry may be advantageous because suchpeptides or proteins may be highly specific biomarkers of a particulardisease, a particular cellular pathway or a particular cell condition.Accordingly, the ability to differentiate between two or more cellpopulations at the single cell level by using peptide, protein or otherbiomarkers, which are analyzable by mass spectrometry, may beadvantageous for one or more reasons, as explained in greater detailbelow.

In one aspect, cell assays featuring mass spectrometric readout areeasily automatable, in particular with respect to the data acquisitionportion of the assay; therefore a large number of cells may be arrayedon a solid support and individually analyzed within a relatively shortperiod of time. For example, as many as 500,000 biological cells, e.g.cultured mammalian cells or drug-exposed mammalian cells or an evenlarger number may be arrayed into individual wells of a 25×75×1 mmmicrowell plate fabricated from a fiber optic bundle, polymer, glass orfused silica and individually measured by MALDI TOF MS or other massspectrometric method. In order to acquire mass spec data from anindividual biological cell, the instrument probing beam should befocused down to an approximately 100 μm diameter or less, preferablydown to approximately 50 μm diameter or less, which is well within thespecifications of the currently available instruments including massspectrometers designed for the tissue imaging measurements. In fact,some instruments e.g. Bruker ULTRAFLEXTREME™ feature laser beam, whichis size tunable down to 20 μm or even down to 10 μm. Depending on aspecific mass spectrometric analytical method, the probing beam may beproduced by an IR or UV laser, or may comprise a stream of charged ions.Note that the diameter of the instrument probing beam in applicationssuch as Matrix-Assisted Laser Desorption Ionization (MALDI), LaserAblation Electrospray Ionization (LAESI), Laser Ablation InductivelyCoupled Plasma (LA-ICP), Secondary Ion Mass Spectrometry (SIMS),Desorption Electrospray Ionization (DESI), nanospray DESI (nano-DESI)and others may exceed dimensions of a single biological cell, in somecases significantly, yet it is still possible to acquire mass spec datafrom a single cell. In an embodiment, this is made possible by providinga microwell array plate, in which individual microwells are sized toaccept a single cell and the adjacent microwells are spaced sufficientlyfar apart from each other, such that a distance between the centers ofadjacent microwells is equal to or greater than the diameter of theinstrument probing beam. For example, individual biological cells thathave linear dimensions of approximately 10 μm may be analyzable bysingle cell WCMS using a MALDI TOF MS instrument equipped with a laserprobing beam focused down to approximately 50 μm when the cells arearrayed on a microwell plate featuring individual microwellsapproximately 15 μm in diameter and spaced apart by approximately 100μm, measured as the distance between the centers of adjacent microwells.Note that in some applications the desorption ionization process iscomplex and may depend on interaction between the probing beam and thecarrier medium, which may be a stream of charged ions in methods such asDESI and LAESI. Accordingly, the instrument performance may becharacterized in terms of spatial resolution rather than the diameter ofthe probing beam. In such cases, which are particularly common in thefield of mass spectrometric imaging, in order to acquire mass spec datafrom a single cell, microwell array plates may be designed to positionadjacent individual biological cells at a distance that is greater thanthe instrument spatial resolution. For example, the nominal spatialresolution of LAESI MS instruments is currently approximately 200 μm.Accordingly, individual biological cells should be positioned at agreater distance from each other, for example 300 μm in order to enableanalysis of single cells using the LAESI technology.

Even when large numbers of cells, e.g. tens of thousands of cells orhundreds of thousands of cells need to be individually analyzed, themass spec data from the entire cell population may be acquiredsufficiently fast, e.g. within several hours provided that theinstrument is configured for such rapid data acquisition. In thisregard, mass spectrometers manufactured by SimulTOF Corporation (SudburyMass.) that feature lasers operating at a frequency of 5 kHz or 20 kHzand therefore capable of acquiring 5,000 or 20,000 single shot spectraper second, may be well-suited for the cell analytical assays disclosedin the instant specification. It is noted that placing individualbiological cells into microwells dispersed on the solid support mayenable fabrication of very dense cell arrays, in which neighboring cellsare positioned sufficiently close to each other to minimize the traveldistance of the instrument probing beam between the successive dataacquisition points, yet remain sufficiently separated on the solidsupport so that spectral data, which is acquired from a specific cell,does not contain spectral contribution from the neighboring cells or atleast spectral contributions from the neighboring cells are minimized.As a non-limiting example, microwell plates fabricated from fiber opticbundles, from photo-structurable glass such as APEX™ glass or from fusedsilica may feature individual microwells separated by as little as 5 μm,which may be measured as a distance between the sidewalls of adjacentmicrowells. The dense packing of microwells may enable overall fasterdata acquisition, while the known parameters of the grid of microwellsmay be used to precisely position the instrument probing beam over thecenter of individual microwells, which may help maximize the signalacquired from individual samples, i.e. from individual cells.

In an embodiment a large cell population, for example a populationcomprising over 1,000 individual biological cells, preferably over10,000 cells, more preferably over 100,000 cells is analyzed on a singlemicrochip using the technique of WCMS performed with sufficiently highspatial resolution. In an embodiment, the methods and devices disclosedin the instant specification enable analysis of a population comprisingapproximately 100,000 biological cells by single cell WCMS in under 24hrs. In an embodiment, a cell population comprising approximately100,000 biological cells is analyzable by single cell WCMS in under 12hrs. In an embodiment, a cell population comprising approximately100,000 biological cells is analyzable by single cell WCMS in under 4hrs. Overall, the amount of time required to analyze a populationcomprising a specific number of cells using the technique of single cellWCMS is determined by multiple factors, some of which are the instrumentthroughput, the distance between adjacent cells positioned on the solidsupport and a number of single-shot spectra, which are averaged in orderto produce a final spectrum. The ability to analyze a large cellpopulation makes it possible to detect rare cells, for example cellsthat constitute less than 10% of the total number of cells within thecell population, preferably less than 1%, more preferably less than0.1%. In an embodiment, the disclosed method of individually analyzingbiological cells by mass spectrometry provides an inexpensive,sensitive, rapid, high-throughput method of detecting rare cell typesthat may be used either as a standalone analytical method or incombination with other known methods of cell analysis, e.g. opticalimaging.

Non-limiting examples of polypeptides and proteins, which may bedetected in a single cell whole cell mass spectrum and therefore may beuseful as molecular indicators of a cell response to a specific stimulusor a specific environmental condition, include ubiquitin, cytochrome C,various histones including histones with various post-translationalmodifications, such as acetylation, methylation, phosphorylation andubiquitination, various defensins, thymosins, various ribosomal proteinsand others. In general, commercially available mass spectrometers, e.g.MALDI TOF mass spectrometers have the detection limit of approximately100,000 molecules of analytes (sub-attomoles), although somestate-of-the-art equipment is capable of detecting even lower numbers.It is therefore possible that analytes present in biological cells inthe amount greater than approximately 100,000 molecules per cell will bedetected at the single-cell level using existing mass spectrometers. Theinstant specification discloses various experimental techniques thatwill help facilitate detection of such low abundance analytes fromindividual cells. In an embodiment, such experimental techniques includeone or more of the following: (i) the ability to position the instrumentprobing (e.g. laser) beam directly over a spot containing the analytesreleased from a single cell, e.g within 5 or less from a center of suchspot thereby eliminating random searching for the optimal signal, whichmay otherwise result in a weaker signal; (ii) the ability to efficientlyrelease analytes from a single cell for the downstream analysis by massspectrometry, including the ability to perform extended (e.g. minutes tohours) incubation of a cell with chemical compounds, which assist in therelease of specific analytes from the cell for the downstream mass specanalysis, such as a digestive compound, a lysis-inducing compound, adetergent compound, etc; (iii) the ability to favorably shape the ionplume produced by the instrument probing (e.g. laser) beam striking theanalyte spot. The latter ability is made possible by positioning a cellinside a microwell, preferably at a specific distance below the topsurface of the microwell plate (e.g. between 10 μm and 50 μm below thesurface), such that the shape of the resulting ion plume is determinedlargely by the sidewalls of the microwell, which surround the analytespot.

The abovementioned polypeptides and proteins, as well as numerous otheranalytes may be also detectable in whole cell mass spectra acquired fromthe samples comprising multiple cells, i.e. not in a single-cell format,as disclosed in greater detail elsewhere in this specification.

It is noted that the majority of current applications of WCMS involvecomparing or identifying different strains of bacteria; theseapplications require detection of only the most prominent peaks in amass spectrum, for example peaks with the relative intensity above the10% or above the 5% threshold. The relative intensity of a peak iscommonly defined as the absolute intensity of that peak divided by theabsolute intensity of the strongest peak present in the same spectrum.Because the experimental conditions of cell culturing may varyconsiderably between different laboratories and even within the samelaboratory, mass spectra acquired from the closely related or even fromthe identical cells cultured at different times will always display somevariability, which is solely due to variations in the cell cultureconditions. Accordingly, there is currently no incentive to detect andanalyze low intensity peaks present in whole cell mass spectra.

Furthermore, analytical methods based on mass spectrometry, inparticular MALDI TOF MS, are rarely used for quantitative detection ofanalytes unless an internal standard is provided. It is well known thatthe intensity of analyte signal in the MALDI TOF mass spectra variesconsiderably between individual single-shot spectra even when thespectra are acquired from the same sample. This effect is ascribed tomultiple factors including a highly inhomogeneous environment resultingfrom the uneven crystallization of the analyte-matrix mixture. Forexample, the presence of “sweet spots”, i.e. areas with a highconcentration of analyte, and consequently, areas that contain little orno analyte is routinely observed in the MALDI TOF MS. The unevendistribution of analyte across the sample spot is encountered even inthe case of samples that contain a single purified analyte. In order tominimize such effects, multiple spectra are usually acquired from randompositions within the sample spot and co-added to produce a finalspectrum.

It could be expected that whole cell mass spectra, which are acquiredfrom extremely non-homogeneous samples, would exhibit even greatervariation of the signal intensity across the sample spot and thus wouldnot be suitable for applications that require quantitative analysis orapplications that require highly reproducible data to be collected intwo or more independent measurements. First, the samples for WCMSanalysis contain thousands of distinct analytes with vastly differentproperties: nucleic acids, lipids, carbohydrates, polypeptides, proteinsand metabolites, among others. Second, the individual analytes are notuniformly distributed throughout a cell, but originate from differentregions within the cell, e.g. an outer membrane, a nucleus, a ribosome,etc, as well as compounds that have been released into the extracellularspace. Therefore, mixing intact biological cells with the MALDI matrixsolution followed by the matrix crystallization is expected to produce asample, in which the spatial distribution of different analytes willfluctuate dramatically across the sample spot.

It is therefore a surprising and unexpected finding that highlyreproducible whole cell MALDI TOF mass spectra may be acquiredconsecutively from a single sample provided that the sample is notsufficiently depleted during such consecutive measurements oralternatively acquired from several samples containing multiple cells.Furthermore, as described below, highly reproducible whole cell massspectra may be acquired by averaging a rather limited number ofsingle-shot spectra collected from a relatively small area on aMALDI-compatible solid support. In an embodiment, the dynamic range ofsignal detection in a whole cell MALDI TOF mass spectrum is 10², i.e.peaks with intensity differing by a factor of 100 may be detected in asingle spectrum. In an embodiment, the dynamic range of signal detectionin a whole cell MALDI TOF mass spectrum is 10³, i.e. peaks withintensity differing by a factor of 1000 may be detected in a singlespectrum. In an embodiment, a peak is considered detected in the massspectrum if its signal-to-noise ratio is at least 3:1, as determined forexample by the industry-standard analytical algorithms. In anembodiment, the position of a specific peak in two independentlyacquired whole cell MALDI TOF mass spectra varies by 50 ppm (parts permillion) or less in the mass range below 10,000 m/z and by 100 ppm orless in the mass range between 10,000-25,000 m/z. The whole cell MALDITOF mass spectra may be independently acquired from a single spot on theMALDI-compatible solid support or alternatively from two or moredistinct, i.e. non-overlapping spots on the solid support. In anembodiment, the relative intensity of a peak at a specific m/z measuredin two independently acquired MALDI TOF whole cell mass spectra variesby 10% or less, preferably by 5% or less, more preferably by 2% or less.The relative intensity of a peak is calculated as the absolute intensityof the peak ratioed against the absolute intensity of the strongest peakpresent in the same mass spectrum. Alternatively, the intensity ratiomay be calculated for any two peaks in a mass spectrum, including peaksseparated by as much as several thousand m/z. In an embodiment, thewhole cell MALDI TOF mass spectra are acquired in the linear positivemode in the 2,000-25,000 m/z mass range. In an embodiment, the wholecell MALDI TOF mass spectra are acquired in the linear negative mode inthe 2,000-25,000 m/z mass range. Alternatively, the whole cell MALDI TOFmass spectra may be acquired in a linear mode below approximately 2,000m/z or in a reflector mode below approximately 3,000 m/z.

In an embodiment, samples for WCMS analysis are prepared by mixingaliquots containing an approximately equal number of biological cellswith a solution of MALDI matrix and allowing the resulting mix to dry indistinct spots on a MALDI-compatible solid support. In an embodiment,the number of cells in such samples, which are fabricated eitherconsecutively or concurrently, differs by less than 50%, preferablydiffers by less than 20%, more preferably differs by less than 10%. Inan embodiment, an absolute intensity of a peak at specific m/z measuredin the whole cell MALDI TOF mass spectra acquired from two or moresamples prepared as described above varies by 20% or less, preferably by10% or less, more preferably by 5% or less. In an embodiment, theabsolute intensity of a peak is calculated after applying one or severalpost-data acquisition spectral processing routines, such as baselinecorrection, spectral smoothing, peak calibration and others.

In an embodiment, a reproducible whole cell mass spectrum is acquiredfrom a sample containing fewer than 10,000 cells, preferably fewer than5,000 cells, more preferably fewer than 1,000 cells. The reproducibilityof a whole cell mass spectrum may be assessed using the quantitativeparameters disclosed in the preceding paragraphs, in particular withrespect to the position of a specific peak within the spectrum, itsspectral width and its absolute or relative intensity. In an embodiment,a reproducible whole cell mass spectrum is acquired by averaging fewerthan 10,000 single-shot spectra, preferably fewer than 5,000 single-shotspectra, more preferably fewer than 2,000 single-shot spectra. Usingstate-of-the-art MS instruments with the ionization laser operating at 1kHz or 5 kHz frequency, the disclosed number of single-shot spectra maybe collected within several seconds or even within several hundredmilliseconds. In an embodiment, a reproducible whole cell mass spectrumis acquired from an area on a solid support that is less than 10 mm²,preferably less than 5 mm², more preferably less than 1 mm². In anembodiment, the extent of analyte depletion in a sample area afterperforming the MS data acquisition is less than 50%, i.e. more than 50%of the analyte remains in the sample area after performing the dataacquisition and is available for the subsequent analysis. In anembodiment, the extent of analyte depletion in a sample area is lessthan 25%, i.e. more than 75% of the analyte remains in the sample areaafter performing the data acquisition.

The disclosed methods, which enable acquisition of highly reproduciblewhole cell mass spectra from a small area on a solid support using alimited number of single-shot spectra, may be particularly advantageouswhen the WCMS data is acquired from cell microarrays or from tissuemicroarrays since microarrays feature a large number of distinct sampleslocalized in very compact microspots, e.g. spots occupying an area thatis smaller than 1 mm². Furthermore, all samples on a microarray areprocessed simultaneously and under identical conditions, which isparticularly advantageous in the case of WCMS analysis.

The disclosed methods of acquiring highly reproducible whole cell massspectra may be also advantageous when analyzing peaks that haveintrinsically low intensity, for example peaks that have 1% relativeintensity compared to the strongest peak in the mass spectrum or evensmaller, for example 0.1% relative intensity. Such peaks maynevertheless reflect a key cellular event or represent a physiologicallyimportant post-translational modification of a protein.

The method of difference mass spectrometry applied to whole cell massspectra

Various methods are known in the art that enable comparison of two ormore mass spectra in order to detect specific differences between thespectra. In the simplest form, two spectra may be presented on acomputer screen and visually inspected for appearance of new peaks andchanges in the intensity of existing peaks. Direct overlay of thespectra and depiction of the spectra in a gel view format, which isalternatively known as a pseudo-gel view, may be used for spectralcomparison, among other techniques. Numerous mathematical algorithmshave been developed for analysis of mass spectra in the digital format,for example by comparing the peak lists.

In an embodiment, there disclosed a method of difference massspectrometry. It has been experimentally established in thisspecification that the whole cell mass spectra acquired from similarsamples (i.e. samples prepared using a similar number of identical ornearly identical cells) using identical instrument settings are highlyreproducible. For example the intensity of individual peaks measured atthe same m/z in such spectra may differ by less than 5%. Accordingly, adifference spectrum may be calculated by subtracting a referencespectrum from a sample spectrum as follows: Difference Spectrum=SampleSpectrum−(Reference Spectrum*C) where C is the subtraction factor. Thesubtraction factor C may be selected to normalize two spectra, in whichthe peaks have different absolute intensity. The spectral subtractionalgorithms are known and incorporated into numerous analytical softwarepackages, for example GRAMS AI Spectroscopy Software available fromThermo Fisher Scientific Inc (Waltham Mass.). Either automated orinteractive spectral subtraction procedure may be performed so that thesubtraction factor C is continuously adjusted in order to minimize theamplitude of individual peaks in the resulting difference spectrum. Inparticular, the least squares method of spectral subtraction may beutilized.

The method of difference WCMS may be used to observe changes in amolecular composition between closely related groups of cells, forexample cells treated with a specific compound versus non-treatedcontrol cells, cells exposed to different concentrations of a specificcompound, cells exposed to different growth conditions, or cellscarrying a mutation or deletion in one or several genes versus thewild-type cells. Importantly, such screenings may be performed in ahigh-throughput fashion in a cell microarray format. It is expected thatthe majority of peaks in the mass spectra recorded from the closelyrelated populations of cells will not change significantly and thus willbe eliminated by the spectral subtraction. Ideally, a difference massspectrum recorded from the identical cell populations resembles a flatbaseline. Peaks appearing in a difference mass spectrum may have eitherpositive or negative value and represent analytes whose concentrationvaries between the two samples. In an embodiment, the spectralsubtraction procedure may be utilized to reveal changes in the low- andmedium-intensity peaks, which would be normally obscured by the presenceof stronger nearby peaks in the original mass spectra.

In an embodiment, the method of difference mass spectrometry is utilizedto establish or confirm the absence of spectral changes and consequentlythe absence of changes in the molecular composition of cells measured intwo or a greater number of samples. For example, a negligible effect ofa particular compound on a specific cell line may be established byrecording mass spectra of the cells treated with such compound versusthe untreated control cells and then comparing the recorded massspectra.

Cell Microarrays Utilizing Microwell Array Plates

In an embodiment, microwell array plates are used as a solid support indevices suitable for tissue culture, cell culture, cell separation, cellenrichment or cell analysis. In an embodiment, the microwell arrayplates of the instant disclosure are configured for providing cellpopulations of a certain size, for example approximately 1 cell,approximately 10 cells, approximately 100 cells, approximately 1,000cells or other number for analysis by one or several analytical methods,for example WCMS.

Microwell plates suitable for use in the microwell cell microarrays maybe fabricated from various materials including unmodified and modifiedsilicon, fused silica, glass, chemically modified glass,photo-structured glass such as APEX™ glass, plastics, polymers, resins,gels, metals and the composite materials. In an embodiment, themicrowell plates of the instant disclosure comprise a fiber optic bundleor a fiber optic faceplate for transmitting an image of a cell array toan output surface.

FIG. 15A depicts a microwell plate-based cell culture and cell analysisdevice according to the embodiment methods of the instant disclosure.The disclosed device comprises a microwell array plate 1501 and aremovable multi-well gasket or a multi-well chamber 1502 reversiblyaffixed to the surface of the microwell plate 1501. The gasket 1502 mayhave self-adhesive properties and may be fabricated from any suitablematerial, for example a biocompatible silicone. Silicone-basedbiomaterials are readily available in various shapes from numerouscommercial vendors. In an embodiment, the gasket material is compatiblewith the sterilization protocols commonly used in cell biology, forexample autoclaving or chemical immersion, e.g. ethanol immersion. Thedisclosed device may further comprise a lid to be placed on top of themulti-well gasket 1502 (lid is not shown in FIG. 15A).

The bottom section of the multi-well gasket 1502 may be dimensioned tomatch the footprint of a standard microscope slide, approximately 25×75mm, or of a standard 384-well microtiter plate, approximately 86×128 mm.Alternatively it may be smaller than these dimensions.

The number of wells, dimensions and shape of individual wells, spacingof wells and the volume of individual wells may be controlled by usingdifferent multi-well gaskets. For example, both square and round shapedwells may be utilized, among other shapes. In an embodiment, thedimensions of individual wells in a multi-well gasket are selected toprovide a sufficient cell growth area, such that the cell populationcultured in a single well may be analyzed with sufficient sensitivity bythe chosen analytical method. For example, round-shaped wells that areapproximately 2 mm in diameter may provide sufficient growth area forvarious types of adherent cells to enable downstream analysis of thecells by WCMS. The wells that are at least several mm in diameter allowmanual or automated dispensing and replacement of used cell growthmedium with fresh medium or with a biologically compatible buffer, e.g.PBS.

Overall, the cell seeding and cell culture methods are similar to thoseused with conventional cell chamber slides. For example, the cells maybe trypsinized and counted, then diluted to a specific concentration,e.g. 5×10⁴ cell/mL, and the cell suspension may be distributed intoindividual wells 1511 of the multi-well gasket, shown in FIG. 15B. Thewells are filled with the cell suspension 1512 in FIG. 15B, covered witha lid and incubated following standard cell culture protocols. The cellculture medium in individual wells may be changed during the cellcultivation. Following the cell culture experiment, the multi-wellgasket may be separated from the microwell array plate, as schematicallydepicted in FIG. 15C.

One of the advantages of using a microwell array plate instead of aflat-surface microscope slide as a solid support for the culture,reaction and subsequent analysis of cells is that a larger number ofcells may be cultured per area unit, e.g per mm² as illustratedschematically in FIGS. 16A-16D, because of the ability of adherent cells1621 to grow on sidewalls 1622 of the microwells 1623 in addition togrowing on the surface between openings into the microwells 1623 and thebottom of the microwells. Depending on the dimensions of individualmicrowells and parameters of the grid formed by the microwells on themicrowell array plate, at least 2-fold and as much as 10-fold greaternumber of adherent cells may be cultured per area unit on athree-dimensional microwell array plate compared to adherent cells 1602cultured on a conventional flat-surface microscope slide 1601. The cellscultured on a microwell plate may grow as a monolayer or alternativelymay grow in several layers thereby forming a three-dimensional cellularmatrix or a tissue microarray. As disclosed previously in thisspecification, the surface properties of a microwell plate may bemodified in a controlled fashion such that the surface area betweenopenings into the microwells may be configured for analysis of cells bymass spectrometry, e.g. may be coated with a layer of electricallyconductive or charge-dissipative material for MALDI MS analysis, whilethe inner surface of individual microwells may be configured for thecell attachment and growth, e.g. may be coated with polylysine or othermaterial with similar relevant properties. The inner surface of amicrowell may be further modified such that the bottom surface will havedifferent surface properties than the sidewalls of the microwell, forexample the bottom surface may be configured for optical analysis ofindividual cells by optical microscopy or fluorescence microscopy. Inaddition, the microwell array plates are also uniquely suitable forculturing non-adherent cells, e.g. cells growing in suspension.

Cells cultured on a microwell array plate both outside and withinindividual microwells 1623 may be readily analyzed directly on themicrowell plate using methods of WCMS. In the case of MALDI MS analysis,application of the MALDI matrix-containing solution to the microwellplate may be achieved by dispensing the solution as an aerosol, bypipetting or by using other known methods including automated liquidhandling systems. The MALDI matrix solution usually contains an organicsolvent, such as acetonitrile, acetone or methanol, which have excellentsurface wetting properties and can readily penetrate into the individualmicrowells 1623 and reach individual cells. The cells inside themicrowells may either detach from the microwell surface or burst uponthe contact with the matrix solution. Upon the solvent evaporation andmatrix crystallization the cell contents become mixed with theionization matrix 1631 and may be localized within the microwell, aswell as the top surface of the microwell plate near the microwell, asillustrated schematically in FIG. 16D. The sample fabricated using thedisclosed procedure may be analyzable by MALDI MS, as well as othermethods of mass spectrometry, e.g. DESI, nano-DESI, LAESI, LMJ-SSP etc.The latter methods of mass spectrometry may be also used for analysis ofcell arrays fabricated using the industry-standard 96-well, 384-well and1536-well microwell plates. Note that the depiction in FIGS. 16B and 16Dis not meant to suggest a specific pattern of cell localization afterthe contact with the MALDI matrix solution but rather a generalillustration of a concept of a re-distribution of analytes released fromindividual cells on a microscope slide or on a microwell array plate,which may occur after the cell lysis. In fact, it is likely that theindividual cells are no longer intact after contact with the matrixsolution followed by matrix crystallization. One potential advantage ofusing the cell lysis and performing the mass spec analysis on thesurface, to which the cells were previously attached is to bypass theneed for the cell separation from the surface, which normally involvesenzymatic digestion, e.g. trypsinization.

In an embodiment, the disclosed combination of a microwell array plateand a multi-well gasket may be utilized in high-throughput screeningapplications aimed at assessing the effect of various compounds on aspecific type of cells, for example to discover novel compounds thatinduce apoptosis. The tested compounds may be isolated from naturalsources or produced by combinatorial synthesis methods or othersynthetic methods and may be supplied in solution, for example in a384-well plate format. The tested compounds may be added along with thecell culture medium to individual wells and their effect on the cellssubsequently measured by WCMS alone or in combination with the opticalimaging of cells. In an embodiment, a flat-surface microscope slide maybe utilized instead of a microwell array plate, as long as the slidesurface is compatible with WCMS and the sufficient number of cells maybe produced for analysis.

In an embodiment, the disclosed combination of a microwell array plateand a multi-well gasket may be utilized to study weakly adherent ornon-adherent cells, for example certain types of bacterial cells, oralternatively adherent cells that become detached from the surface ofthe microwell plate, for example as a result of cell death. In suchcase, the suspended cells may be precipitated to the surface of themicrowell plate by centrifugation of a combination of the microwellplate and the multi-well gasket at approximately 2,000 rpm or othersuitable speed. This procedure may be also utilized to rinse the cellsdirectly on the microwell plate, i.e. to replace the cell growth mediumwith an appropriate biological buffer, such as PBS prior to the WCMSanalysis.

Furthermore, in contrast to the conventional microscope slides, whichpossess smooth top surface and therefore unable to retain non-adherentcells, the microwell array plates of the instant disclosure are capableof retaining non-adherent cells on the plate within individualmicrowells. This feature provides substantial advantage because thenon-adherent cells may be cultured, reacted with specific compounds andanalyzed directly on the microwell plate, for example by WCMS. Thedimensions of individual microwells may be selected to accommodate aspecific number of cells, e.g. approximately 10 cells, approximately 100cells, approximately 1,000 cells or other number in order to achievesufficient sensitivity of the downstream analytical method. Thus, themicrowell plates are suitable for fabrication of live cell arrays.

Microwell Cell Microarrays Utilizing Microbeads

In an embodiment, the microwell array plates of the instant disclosureare utilized in combination with a plurality of microbeads to createcomposite microbead-microwell microarrays suitable for the cell culture,cell separation, cell enrichment and cell analysis applications. In anembodiment, the disclosed composite microbead-microwell microarrays areconfigured for high-throughput screening applications, for examplescreening of hundreds or even thousands of different compounds on asingle microchip. In an embodiment, the disclosed composite microarraysare utilized for cultivation of cells obtained from a clinical specimen.In an embodiment, individual microbeads of the instant disclosure areconfigured for providing cell populations of a certain size, for exampleapproximately 1 cell, approximately 10 cells, approximately 100 cells,approximately 1,000 cells or other number for the downstream analysis bymass spectrometry. In an embodiment, individual microbeads of theinstant disclosure are configured for releasing compounds from the beadsfor the subsequent uptake by cells in the cell microarray.

FIG. 17A schematically shows cross-section of a small region of anembodiment composite microbead-microwell microarray. In reference toFIG. 17A, the microarray may comprise a microwell plate 1701 and aplurality of microbeads 1705 distributed into individual microwells1702. Individual microwells 1702 may be further separated from eachother by means of optional walls 1703 provided, for example, by amulti-well gasket affixed to the top surface of the microwell plate1701. The walls 1703 may serve to separate individual microwells or toseparate groups comprising multiple microwells. The microwell plates maybe fabricated from fused silica, glass, plastics, gels, metals andcomposite materials. Individual microbeads may be fabricated from anysuitable material including silica, glass, hydrogel, polymers, resinsand composite materials. In particular, beads used in the combinatorialsynthesis including one bead-one compound (OBOC) and one bead-twocompound (OB2C) combinatorial libraries may be utilized. A non-limitingexample of commercially available beads, which are suitable forfabricating a microarray of the present disclosure, is TENTAGEL™ resinwith 90 micron particle size. In an embodiment, a bead possesses asurface that is sufficiently smooth, sufficiently non-porous (e.g. thesize of pores may be less than 100 nm) and sufficiently hydrophilic.Non-limiting examples of such beads are borosilicate and sold-lime glassbeads and plain silica microspheres, all of which are available fromPolysciences, Inc (Warrington, Pa.) and other vendors. One potentialbenefit of using glass or silica beads in conjunction with the cellculture assays is that such beads may be placed in contact with abiological cell culture medium for an extended period of time (e.g.days) without depleting compounds present in the culture medium, whichoccurs more readily when the polymer microspheres are utilized, eitherthrough adsorption on the polymer bead surface or absorption within thepolymer bead core.

Individual microbeads 1705 may be conjugated to active agents 1706 thatare potentially capable of eliciting a specific cell response, forexample cell binding, cell division, cell signaling, expression of aspecific protein, cell death etc. The active agents 1706 may comprisepolypeptides, proteins, protein complexes, carbohydrates, lipids,nucleic acids, small molecules, hormones, signaling molecules,pharmaceutical compounds, etc. The individual microbeads 1705 may beencoded using positional encoding, optical encoding or mass tag encodingincluding mass tags localized inside the topologically segregatedbilayer beads. In an embodiment, the beads are not encoded by theconventional means of bead encoding and the active agents 1706 arereleased from the beads and identified by mass spectrometry on themicrowell plate 1701.

An exemplary method of utilizing the composite microbead-microwellmicroarrays of the instant disclosure is schematically depicted in FIGS.17A-17C. In reference to FIG. 17A, individual microwells 1702 containingmicrobeads 1705 conjugated to active agents 1706 may be filled with acell seeding medium 1707 containing a suspension of cells 1708 atspecific concentration, e.g. 1×10³ cells per mL. In reference to FIG.17B, active agents 1706 may be subsequently released from the beads 1705and mixed with the cell medium 1716, which may be achieved by passivediffusion. In an embodiment, the active agents 1706 are conjugated tothe beads 1705 via a UV photolabile linker and released from the beadsby exposing the composite microbead-microwell microarray of the instantdisclosure to a light of specific wavelength, e.g. near 365 nm.Commercially available glass microwell plates, e.g. plates manufacturedfrom APEX™ glass or from fused silica have sufficiently hightransmission in the spectral region around 365 nm to enable rapid, e.g.within 5 min, photorelease of a substantial fraction of a peptide,protein, small molecule or other compound, which is conjugated to glass,TENTAGEL™ or another type of beads via commonly used UV-photosensitivelinkers, by UV light delivered to the beads positioned inside themicrowells through the bottom of the microwell plate. In fact, over 90%of a bead-bound compound may be released within 5 min of UV exposurefrom a bead positioned on a microwell plate using the disclosed method.In an embodiment, the photorelease reaction is performed with highspatial resolution from inside the individual microwells for example byutilizing microwell array plates fabricated from a fiber optic bundle,such that a UV source is functionally connected to the individualmicrowells by means of one or several optic fibers. The ability torelease the active agents 1706 from the beads 1705 by photolysis of aphotosensitive linker also enables various forms of kinetic assaysbecause of the ability to trigger the release of an active agent atspecific time points. By providing the microbeads with known bindingcapacity and known reaction volume, which is defined by the knowndimensions of the microwell plate, such as depicted in FIG. 17A, it ispossible to provide precisely measured concentration of an active agentreleased into the cell culture medium 1716, for example 1 nM, 100 nM, 1μM etc.

In an embodiment, the active agents 1706 may remain conjugated to thebeads 1705, i.e. the cells may react with an active agent, which isimmobilized on the outer surface of a bead. In an embodiment, asufficiently long spacer between the bead and the active agent mayfacilitate reaction between the active agent and the cell localizedsufficiently close to the bead surface.

In reference to FIG. 17C, the cells may be grown to a confluence or to alower density in a presence of the active agents released from thebeads. Adherent cells may be bound to the top surface of the microwellplate 1721, between openings into the microwells, to the sidewalls ofthe microwells 1722, to the bottom of the microwells 1723, to the outersurface of the beads 1724 or all of the above. The cells may besubsequently analyzed directly on the microwell plate by variousanalytical methods including optical spectroscopy and mass spectrometry.

The disclosed methods and compositions are compatible with various knowntechniques of cell labeling including labeling with fluorescent markersand stable isotope labeling.

Several data readout options, which are possible with the compositemicrobead-microwell microarrays of the instant disclosure, will befurther explained with reference to FIGS. 18A-18C. FIGS. 18A-18Cschematically depict a single microwell 1801 within a microwell arrayplate with a bead 1802 placed inside the microwell. In reference to FIG.18A, biological cells 1804 located on the top surface of microwell platebetween openings into the microwells and biological cells 1805 locatedon the surface of the beads may be analyzed by optical methods from thetop surface of the microwell plate, as exemplified by an UP arrow.Microwell plates fabricated from optically transparent materials, e.g.glass also enable analysis of the cells 1803 on the microwell plate byoptical methods performed from the bottom surface of the microwellplate, as exemplified by a DOWN arrow. The optical methods of analysismay comprise visible microscopy including live cell microscopy andfluorescence microscopy. Furthermore, the optical methods of analysismay comprise cell counting, measurement of the cell density, measurementof the cell morphology and detection of localization of specificbiomolecule targets within the cell by immunofluorescence. With respectto the latter, all necessary steps including cell fixation, cellpermeabilization, cell staining and cell washing may be performeddirectly on the microbead-microwell microarray. In reference to FIG.18B, the cells may be analyzed by mass spectrometry, for example usingthe method of WCMS, from the top surface of the microwell plate asexemplified by an UP arrow. The mass spectrometric analysis may compriseMALDI, DESI, nanoDESI or other suitable methods of analyte ionization.The mass spectrometric analysis may comprise time-of-flight, FT-MS, iontrap, quadrupole, tandem MS, hybrid MS and other known methods. Inreference to FIG. 18C, individual beads 1821 that possess opticalencoding or encoding by mass tags may be identified by optical and massspectrometric methods, respectively, directly on the microwell arrayplate. In an embodiment, optical or mass spectrometric analysis of thebeads located on the microwell plate is performed concurrently withanalysis of the cells located on the microwell plate.

The composite microbead-microwell microarrays may be utilized in variousalternative embodiments. For example, the cells may be grown on thesurface of beads, which are suspended in a cell growth medium inside aflask or a similar suitable vessel. The cells may be grown to confluenceor to a specific density, at which point the beads with the attachedcells may be transferred on a microwell array plate and distributed intoindividual microwells at one bead per well occupancy using thepreviously disclosed methods.

The disclosed method of distributing microbeads with cells bound theretointo individual microwells of a microwell array plate and subsequentlyanalyzing the cells by WCMS may be particularly suitable forapplications involving purification and analysis of rare cells obtainedfrom a complex biological source, e.g. blood. For example, the disclosedmethod is suitable for isolation and characterization of circulatingtumor cells (CTCs) that is cells, which originate in a primary or ametastatic cancer and are released into the bloodstream. FIGS. 19 A-Fschematically depict a method of capturing and analyzing cells using acomposite microbead-microwell microarray of the instant disclosure. Inreference to FIG. 19A, an individual microbead 1901 is conjugated to anaffinity capture reagent, e.g. an antibody 1902, multiple identicalcopies of which may be present on the bead. For example, beads that areconjugated to an antibody against epithelial cell adhesion molecule(EpCAM) may be used to capture CTCs expressing EpCAM from the clinicalblood specimens. Note that the diameter of the microbead 1901 determinesthe maximum number of cells, which can be captured on a single bead,i.e. the smaller beads will capture fewer cells. The microbead 1901 mayalso have distinctive optical, e.g. fluorescent properties, which willfacilitate subsequent detection of the microbead position on themicrowell plate. The microbead 1901 may or may not have magneticproperties. In reference to FIG. 19B, biological cells 1911 may becaptured on the antibody-conjugated bead 1912 using a microfluidicdevice such as a microcapillary 1914 or alternatively using a suspensionof beads in a liquid medium. One or more beads with captured cells 1924are then arrayed on a microwell plate 1921 inside individual microwells1922 using previously disclosed methods, as schematically shown in FIGS.19C and 19D. The captured cells are mixed with a MALDI ionization matrix1932 directly inside the microwells 1922, as shown in FIGS. 19E and 19Fand subsequently measured by mass spectrometry.

The disclosed method may provide one or several advantages disclosedbelow and is particularly suitable for the high-sensitivity measurementby mass spectrometry when only a limited number of cells are availablefor analysis. First, the beads with the bound cells are placed intomicrowells, which are arranged in an ordered grid on a microwell plate.Because the parameters of the grid of microwells are known, it ispossible to place the ionization laser beam of the mass spectrometer1942 very close, e.g. within several microns from the center of amicrowell in which the bead 1924 is located, thereby eliminating theneed to randomly search for the strong analyte signal. Furthermore, thecells are mixed with the MALDI matrix in a volume, which is defined bythe dimensions of a microwell. As a result, the analyte migration on themicrowell plate is limited to a single microwell. Accordingly, highlyconcentrated analyte spots as small as 40 μm in diameter or less may beproduced using the disclosed approach, which enables acquisition ofhigh-quality whole cell mass spectra 1946. In an embodiment, theanalytes, which are mixed with the ionization matrix, remain localizedentirely within a microwell and below the top surface of the microwellplate. As disclosed in the U.S. Pat. No. 7,695,978, the sidewalls of amicrowell function to shape the ions generated by the laser striking thesample into a tightly focused ion beam thereby further significantlyimproving the analyte detection sensitivity.

In an embodiment, the disclosed composite microbead-microwellmicroarrays enable analysis of a cell response against a combination ofat least two distinct active agents, which are delivered to themicroarray via microbeads positioned inside individual microwells on amicrowell plate and the wells of a multi-well gasket affixed to the topsurface of the microwell plate, respectively. FIG. 20 schematicallydepicts a section of a composite microbead-microwell microarrayaccording to the instant specification, which comprises a microwellarray plate 2001 and a multi-well gasket 2002 affixed to the top surfaceof the microwell plate. Individual microwells 2003 of a microwell plate2001 may contain one or several beads with active agents conjugatedthereto, as disclosed previously. Separately, individual wells of amulti-well gasket 2002 may be filled with a liquid medium 2004containing an additional distinct active agent, which is mixed with theliquid medium. Accordingly, the cells within such microarray may besimultaneously exposed to two or more active agents, one of which may beidentical throughout the microarray area defined by a well of themulti-well gasket 2002. The adjacent wells 2005 are fluidicallydisconnected from each other by means of walls 2006 provided by themulti-well gasket 2002 and therefore may contain different active agentsor a same active agent in different concentrations. Active agentsdelivered via microbeads positioned inside the microwells may bereleasable from their carrier microbeads, for example by photolysis of aUV-sensitive linkage or alternatively may remain conjugated to themicrobeads. In an embodiment, the active agents bound to the carriermicrobeads comprise a cDNA library or an RNAi library suitable for acell transfection assay.

In an embodiment, the disclosed devices, methods and compositions may beutilized to study of processes of cell division, cell migration andother related biological phenomena including cell invasion andchemotaxis. In particular, the methods disclosed in the instantspecification enable mass spectrometric detection of biological cells onthe surface of the solid support performed in a high spatial resolutionmode, e.g. with lateral resolution of 50 micron or better. Therefore,the methods of whole cell mass spectrometry including MALDI TOF MS,which are disclosed in the instant specification, may be useful in acell migration assay, cell invasion assay, cell chemotaxis assay andother related functional assays either as a sole readout tool or incombination with other methods, such as optical imaging, fluorescenceimaging, infrared spectroscopy, Raman spectroscopy, Surface PlasmonResonance (SPR) etc.

In an embodiment, the instant specification discloses reactive cellarrays comprising one or more different cell types, which are suitablefor screening libraries of bead-conjugated compounds for biologicalactivity. In reference to FIG. 21A a reactive cell array may comprise amicrowell array plate 2101 and a layer of live biological cells grown ona surface of the microwell array plate. The cells may be present on thebottom surface of individual microwells 2102, on the sidewalls ofindividual microwells 2103 and on the top surface of the microwell plate2104. The cell array may further include a liquid medium 2105 in contactwith the surface of the microwell plate. The liquid medium may be a cellculture medium or a cell growth medium. The cell array may furtherinclude a gasket affixed to the top surface of the microwell plate andan optional lid. The gasket may serve to separate areas within themicrowell plate that contain different cell types. The cell array may bekept at conditions compatible with the survival and growth of a specificcell type, e.g. sterile environment, optimal temperature (near 37° C.),suitable concentration of CO₂, specific compositions of the cell growthmedium, etc. The cells may be grown to approximately 50% confluency,although either lower or higher confluency may be also acceptable. Cellspresent within such reactive cell array may remain viable for severaldays and furthermore the cell array may be packaged and shipped usingprecautions normally associated with shipping samples comprising livebiological cells. In an embodiment, the liquid medium 2105 containsglycerol or other suitable compound and the cell array may be storedand/or shipped at sufficiently low temperatures, e.g. approximately −20°C. or approximately −80° C. Examples of producing and analyzing thereactive cell arrays are provided in the EXAMPLES section.

In reference to FIG. 21B, the reactive cell array has several featuresthat enable its use in screening of a library of bead-conjugatedcompounds, which include the following: (i) the cell array contains anarray of microwells, which are size-tuned for accepting size-matchingmicrobeads 2111 at one bead per well occupancy. For example 250 μmdiameter microwells with an approximately 20 μm thick surface layer ofbiological cells will accept beads that have diameter of about 150 μm,about 175 μm, or about 200 μm at one bead per well occupancy; (ii) themicrowell plate may be fabricated from an optically transparentmaterial, e.g. fused silica or may contain optic fibers, which willenable photorelease of the bead-conjugated compounds via photolysis of aphotolabile linker while the beads are positioned inside the microwells.Other methods of the compound release from the beads may becontemplated; (iii) the microwell plate enables acquisition of massspectrometric and optical, e g. fluorescence data from both the beadsand the cells.

In reference to FIG. 21C, an active compound may be released from a beadpositioned inside a microwell such that the released compound 2121 islocalized predominantly within the corresponding microwell and iscapable of reaching the cells localized on the inner surface of themicrowell, e.g. via diffusion in the liquid medium. The distance betweenthe bead surface and the cells localized on the surface of a microwellmay be from less than 1 μm to approximately 100 μm or greater. Multiplecompounds may be simultaneously released from multiple beads within thearray of beads. The cell array may be incubated with the bead-releasedcompounds for an extended amount of time, e.g. 30 min, 1 hr, 6 hrs, 12hrs, 24 hrs, 2 days etc.

In reference to FIG. 21D, following incubation of the cell array withthe compounds released from the beads, the reacted cell array may beanalyzed by one or more analytical methods including mass spectrometryand fluorescence. The cells 2131 that have been exposed to abiologically active compound may be compared to the un-exposed controlcells 2132 in order to detect changes in the cell mass spectral profile,cell morphology, localization of specific analytes within the cells byimmunostaining etc. Note that cells 2133 that detach from the sidewallsof the microwells may still be localized predominantly within theircorresponding microwells because of the three-dimensional structure ofthe solid support 2101.

The present disclosure is described in the following Examples, which areset forth to aid in the understanding of the disclosure, and should notbe construed to limit in any way the scope of the disclosure as definedin the claims which follow thereafter. The following examples are putforth so as to provide those of ordinary skill in the art with acomplete disclosure and description of how to make and use the presentdisclosure, and are not intended to limit the scope of the presentdisclosure nor are they intended to represent that the experiments beloware all or the only experiments performed. Efforts have been made toensure accuracy with respect to numbers used (e.g. amounts, temperature,volume, time etc.) but some experimental errors and deviations should beaccounted for.

EXAMPLES

Materials and Methods

Microwell array plates were fabricated by INCOM Inc (Charlton Mass.) inseveral configurations: (1) 75 mm×25 mm Rectangular Fiberoptic FaceplatePlano/Plano w/Corner Chamfers and Side Bevels 75.0 mm×25.0 mm×1.0 mmThick. Material: Block Press BXI84-50 with interstitial EMA. NA 1.0, 50micron fiber size. One side is etched to 30 micron depth; (2) same asabove, except that one side is etched to 55 micron depth; (3) 79 mmRectangular Fiberoptic Faceplate w/Magenta SU-8 Coating, Plano/Plano,75.00 mm×25.00 mm×1.00 mm Thick. Material: Block Press BXI87-6 withinterstitial EMA. NA 1.0, 6 micron fiber size. Polymer Coating, SquarePack. Well Diameter: 180 micron. Well Pitch: 200 micron. CoatingThickness: 200 micron.

Microwell array plates were fabricated by Trianja Technologies Inc(Allen Tex.) in several configurations: (1) APEX™ Glass MicroscopeSlides with Customized Microwell Array, 200-250 micron diameter wells,180±25 micron depth, 500 micron pitch; (2) same as above except that thetop surface of the slides was coated with a 5-10 nm thick layer ofchrome; (3) APEX™ Glass Microscope Slides with Customized MicrowellArray, 200-240 micron diameter wells, 100±10 micron depth, 500 micronpitch; (4) APEX™ Glass Microscope Slides with Customized MicrowellArray, 100-140 micron diameter wells, 100±10 micron depth, 500 micronpitch.

The ProPlate™ Slide Chamber System for the fabrication of beadmicroarray was from Grace™ Bio-Labs (Bend Oreg.). The multi-arraychamber set included Tray and Cover, four 1-well Proplate modules,Delrin Snap Clips and seal strips.

The removable multi-well silicone gasket used in a cell cultivationchamber was from Ibidi LLC (Verona Wis.). The self-adhesive siliconegasket contained 12 wells, each well was 7.5×7.5×8 mm measured aswidth×length×height, holding approximately 250 μL volume of a liquidmedium.

The bead-conjugated peptides were custom synthesized by 21^(st) CenturyBiochemicals Inc (Marlborough Mass.). General structure of thepeptide-bead constructs was [PEPTIDE]-[GGGSGGSG]-[PLL]-[PEG]-[TG Bead](SEQ ID NO: 25) where [PEPTIDE] is a variable peptide sequence givenbelow, [GGGSGGSG] (SEQ ID NO: 1) is a flexible Glycine-Serine spacer,[PLL] is a 365 nm Fmoc photolabile linker structurally similar to thecatalog item RT1095 from Advanced ChemTech (Louisville Ky.), [PEG] is apoly-PEG spacer at least 2 units long and [TG Bead] is a 90 microndiameter TentaGel™ resin available from Rapp Polymere (Tubingen,Germany).

The ten variable peptide sequences, written in the N-terminus toC-terminus notation, were: PPGFSPFR (SEQ ID NO: 2), RPPGFSPFR (SEQ IDNO: 3), RPPGFSFFR (SEQ ID NO: 4), RPPGFSRFR (SEQ ID NO: 5), ISRPPGFSPFR(SEQ ID NO: 6), WQPPRARI (SEQ ID NO: 7), APRLRFYSL (SEQ ID NO: 8),TRNYYVRAVL (SEQ ID NO: 9), KQPELAPEDPED (SEQ ID NO: 10), YTDIEMNRLGK(SEQ ID NO: 11).

An exemplary procedure utilized by the manufacturer in the peptidesynthesis protocol is given below. An acid resistant PEG amide resin(Rapp Polymere), was used for peptide manufacture (scale of 5 micromolesper peptide) to allow for the isolation of peptides free from side-chainprotected but still attached to the resin. The N-alpha-Fmoc andside-chain protected L-amino acids were dissolved in DMF and activatedusing HATU (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate) and for a double coupling HCTU[O-(1H-6-Chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate]. The Fmoc-protected photolabile linker(4-{4-[1-(9-Fluorenylmethyloxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy}butanoic acid) was activated using HATU, at which time that step and allsubsequent steps were performed in extremely low light. The activatedamino acid was added at a 4-fold excess to peptide resin. An 8-foldexcess of DIPEA (N,N-diisopropylethylamine) was added and the reactionproceeded for 40-80 minutes at room temperature. Once peptide synthesiswas complete, the N-terminal Fmoc group was removed to uncover theN-terminal amine and the resin subjected to cleavage inTFA:triisopropylsilane:water (95:2.5:2.5, v/v/v). The resins were washedwith neat TFA and then DCM 5 times and dried.

The bead-conjugated fluorescent peptide[FL]-[GKGEAIYAAPFAKKK]-[GGGSGGGG]-[PLL]-[PEG]-[TG Bead] (SEQ ID NO: 12),written in the N-terminus to C-terminus notation, where [FL] is aisothiocyanate derivative of fluorescein with molecular weight of 389 Dawas custom synthesized using the standard methods of Fmoc chemistry.

A TRNYYVRAVLGGGSGGSG peptide (SEQ ID NO: 13) was purified toapproximately 95% purity by HPLC and subsequently conjugated to 170 μmdiameter TentaGel resins that additionally contained a 365 nmphotolabile linker.

All enzymes and solvents were from Sigma-Aldrich (St. Louis Mo.).

Experimental Results

Some of the experiments performed using the compositions and methodsdisclosed in this application and the resulting experimental data aredescribed below.

Example 1

Assembling a Bead Library Suitable for Fabrication of a Reactive BeadMicroarray

The ten peptide-bead conjugates were suspended in deionized H₂O andstored individually in a lightproof container at 4° C. or alternativelyin a 30% (v/v) glycerol solution at −20° C. Bead suspensions containingapproximately 100 beads of each type were combined in a 1.5 mL plasticmicrocentrifuge tube and immediately used for the microarraypreparation.

Example 2

Fabrication of a Reactive Bead Microarray Using a Microwell Array Plate

A 25×75×1 mm microwell array plate subdivided into 16 sub-array regions,each sub-array region measuring 6 mm×6 mm and containing 169 microwellswas inserted into a ProPlate™ slide chamber module featuring 16size-matching (7 mm×7 mm) chambers and secured within the slide chambermodule using Delrin snap clips. Approximately 250 μL of mass spec gradedeionized H₂O was added into each slide chamber and the ProPlateassembly was centrifuged on Eppendorf® 5804R centrifuge equipped with amicrotiter plate adapter at 2,500 rpm for 15 min to fill the individualmicrowells with water. In a separate experiment, the ProPlate assemblywas placed under vacuum prior to the centrifugation step in order torelease the air bubbles trapped inside individual microwells.

A bead suspension containing approximately one thousandpeptide-conjugated microbeads in mass spec grade deionized H₂O wasdispensed into an individual slide chamber on the ProPlate assembly andthe beads were randomly distributed into individual microwells byplacing the entire assembly on a laboratory nutator for 30 min. Thebeads were protected from the ambient light by covering the nutator withaluminum foil. The ProPlate assembly was again centrifuged at 2,500 rpmfor 15 min and then disassembled by removing the Delrin snap clips torelease the microwell plate.

The microwell plate was then submerged into a container filled with 100mL of mass spec grade deionized H₂O and gently shaken for 5 min toremove loose beads, i.e. the beads that did not sink into themicrowells, from the surface of the microwell plate. Alternatively, themicrowell plate was gently rinsed under a stream of deionized H₂O. Themicrowell plate with beads placed inside the microwells was air-driedand stored inside a lightproof container at −20° C.

The described method enables facile fabrication of random beadmicroarrays. Because multiple beads are simultaneously placed on themicrowell plate surface and distributed into individual microwells,microarrays featuring hundreds of thousands of reactive sites may berapidly produced.

Positioning of the beads into the individual microwells was confirmed byexamining the microwell plate using an inverted microscope (Nikon®Eclipse TS100). One bead per microwell occupancy was observed for 170 μmbeads placed inside the 200 μm wide/200 μm deep microwells and for 90 μmbeads placed inside the 100 μm wide/100 μm deep microwells. Greater thanone bead per microwell occupancy, e.g. 2-3 beads per microwell wasobserved for 90 μm beads placed inside the 200 μm wide/200 μm deepmicrowells. In the latter case, however, loading a sufficiently smallnumber of beads also results in the one bead per microwell occupancyeven though the microwells can accommodate more than one microbead. Arepresentative microphotograph of a bead placed into a microwell on amicrowell plate is shown in FIG. 22.

Example 3

Contacting a Microbead Array with a Sample Possessing an EnzymaticActivity

A reactive bead microarray was fabricated as described in the previousExample. The microarray contained 10 distinct bead-conjugated peptidesubstrates randomly arrayed in multiple replicates in 169 spots forminga square grid within a 6×6 mm area. The beads were placed eitherapproximately 10 μm or approximately 100 μm below the surface of themicrowell plate by selecting an appropriate combination of a beaddiameter and the microwell depth. The individual peptide substrates wereselected to contain several protease-sensitive bonds, therefore thefabricated microbead array was suitable for detecting a proteolyticactivity in a sample and more generally, was suitable for proteaseprofiling studies. The samples contacted with the microarray were: (1)an aqueous solution of bovine trypsin at the concentration of 30 μg/mL;(2) an aqueous solution of thermolysin from Bacillus thermoproteolyticusrokko at the concentration of 50 μg/mL and (3) an aqueous solution ofproteinase K from Tritirachium album at the concentration of 50 μg/mL.The samples were prepared by reconstituting a lyophilized powdercontaining the enzyme in mass spec grade deionized H₂O.

The samples were contacted with the reactive sites of the microarrayusing several methods.

Method 1: approximately 100 μL of an enzyme solution was placed on asurface of the microarray to cover the entire 6×6 mm area and allowed tospread on the microwell plate to reach the microarray reactive sites(beads), which were located approximately 10 μm below the surface of themicrowell plate.

Method 2: approximately 200 μL of an enzyme solution was dispensed tocover the 6×6 mm area of the microarray, in which the reactive siteswere located approximately 100 μm below the surface of the microwellplate. The microarray was then placed under vacuum to release airbubbles trapped inside the microwells and consequently to allow thesample solution to enter individual microwells and reach the reactivesites. The excess sample solution remaining on the surface was manuallywithdrawn by pipetting, so that the sample presence would be restrictedto individual microwells.

Method 3: the reactive sites were located either 10 μm or 100 μm belowthe surface of the microarray plate. The sample was contacted with themicroarray in the form of an aerosol generated by an Aztec airbrush(Testors Corp, Rockford Ill.). Approximately 5 mL of enzyme solution wasloaded into an airbrush sample cup and the microarray plate was exposedto a stream of sample-containing microdroplets generated by theairbrush, which was achieved by placing the plate approximately 10 cmfrom the airbrush nozzle at a 90 degree angle to the direction of thestream. The duration of the sample application to the microarray waslimited to approximately 1 minute to achieve delivery of a sufficientamount of sample but avoid merging of individual droplets into muchlarger spots on the microarray surface.

Microarrays contacted with a sample using methods 1, 2 and 3 wereincubated for at least 2 hrs at 37° C. in a humidified container. Insome cases, the microarrays were incubated with a sample overnight.

Example 4

Release of an Active Agent from a Microarray Reactive Site

Active agents, which in this example are the peptide substratesconjugated to their carrier microbeads via a photolabile linker, werearrayed on a microwell plate as described previously. The microwellplate was placed within 5 cm from a 365 nm UV source (Lamp Black-Ray VLVVL-21 CSA, Utech Products, Schenectady N.Y.), such that the UV lightwas delivered either via the bottom of the microwell plate through theUV-transparent glass core of the plate or from the top of the microwellplate through openings into the microwells. Dry beads arrayed on themicrowell plate were UV irradiated for 15 to 30 mins. In someexperiments the UV irradiation reaction was performed on beads submergedinto microwells filled with either deionized H₂O or 1% solution ofglycerol.

Example 5

Solid MALDI Matrix Suitable for Mass Spec Imaging of a Microbead Array

Ultrapure CHCA matrix was purchased from CovaChem (Loves Park, Ill.).Approximately 100 mg of matrix was placed in a porcelain mortar andground with a pestle until the mixture appeared homogenous. The groundmatrix was suspended in 1 mL of mass spec grade deionized H₂O andtransferred into a 1.5 mL plastic Eppendorf® microtube. The largercrystals settled at the bottom of the microtube within 1 min. Theparticles that remained in suspension after 1 min were transferred intoa separate tube and used in the microarray mass spec imagingexperiments.

FIG. 23 is a microphotograph of CHCA matrix crystals prepared using thedescribed grinding and sedimentation method. The generally uniformmicroparticles appear to be approximately 10 μm in diameter.

Example 6

Application of MALDI Matrix to a Microbead Array

Microbeads conjugated to the peptide substrates via a photolabile linkerwere arrayed on a microwell plate as described previously. Themicrobeads were placed approximately 100 μm below the surface of theplate by selecting an appropriate combination of the bead diameter andmicrowell depth. The microwell plate was then inserted into the ProPlateslide chamber module and an aqueous suspension of CHCA matrixmicrocrystals, which was prepared as described in the previous Example,was added into individual chambers in the amount sufficient tocompletely cover the surface area within the individual chambers. Theentire assembly was centrifuged in a microtiter plate adapter onEppendorf® 5804R centrifuge at 2,500 rpm for 15 min in order to placethe matrix microcrystals inside the microwells. The microwell plate wassubsequently removed from the ProPlate module and gently rinsed under astream of deionized H₂O to remove the loose microcrystals of matrixpresent on the surface of the microwell plate between the microwells.This procedure restricted the presence of matrix to individualmicrowells.

The microwell plate was subsequently air-dried and the beads inside themicrowells were exposed to the 365 nm UV light delivered through thebottom of the microwell plate as described previously. The duration ofthe UV exposure was between 15 and 30 min. The microwell plate was thenplaced in a sealed humidified container and incubated overnight at roomtemperature to enhance adsorption of the photoeluted peptide analytes tothe matrix crystals. In a separate experiment, the UV photoelutionreaction was performed on the bead-matrix mixture submerged intomicrowells filled with deionized H₂O, i.e. prior to drying. A microscopeglass coverslip placed on top of the microwell plate was used to preventwater evaporation during the photoelution procedure.

Example 7

Application of MALDI Matrix to a Microbead Array

This experiment was performed to determine experimental conditions thatallow uniform MALDI matrix coverage of a three-dimensional surface of amicrowell array plate including top surface of the plate and bottomsurface of individual microwells.

The 10 mg/mL solution of CHCA matrix in 50% acetonitrile and 0.2% TFAwas prepared as described previously. A microbead array was fabricatedby positioning 75 diameter beads on a microwell plate featuring 200 μmdeep and 200 μm wide microwells.

The microbead array was coated with CHCA matrix delivered as an aerosolgenerated by an Aztec airbrush (Testors Corp, Rockford Ill.).Approximately 5 mL of the matrix solution was loaded into an airbrushsample cup and the microarray plate was exposed to a stream ofmatrix-containing microdroplets generated by the airbrush, which wasachieved by placing the plate approximately 10 cm from the airbrushnozzle at a 90 degree angle to the direction of the stream. The durationof the matrix solution application to the microarray was 20 secondsfollowed by 1 min of air exposure to allow the solution to air dry. Thematrix application cycle was repeated 2 more times. Only a limitedamount of CHCA matrix was applied to the microwell plate using thedescribed procedure in order to produce spatially resolved spotscontaining the crystallized matrix. In order to achieve completecoverage of the surface, several additional cycles of the matrixapplication would be required.

Localization of the individual matrix spots generated by the proceduredescribed above was determined by inspecting the microwell plate usingan inverted microscope (Nikon® Eclipse TS100). FIGS. 24A-24B show imagesof the microwell plate captured at different focus distance, namelyfocusing on the surface of the plate (FIG. 24A) and on the bottom of themicrowells (FIG. 24B). It is noted that the above-described method ofmatrix application enables delivery of the matrix simultaneously at thesurface of the microwell plate and into individual microwells;furthermore dimensions of the individual matrix spots produced at thesurface of the microwell plate and at the bottom of individualmicrowells are approximately equal. In addition to the airbrush, thedescribed method may be used with the other droplet-generatingequipment, namely a TLC sprayer, a nebulizer, a specially designedmatrix spray robot, etc.

Example 8

Simultaneous Imaging of a Microwell Array Plate, an Array of Microbeadsand an Array of Analytes Released from the Microbeads Using MALDI TOF MS

An array of microbeads conjugated to a peptide TRNYYVRAVLGGGSGGSG (SEQID NO: 13) via a photolabile linker was fabricated on the APEX™ glassmicrowell plate using the previously described methods. The bead arrayfabricated in this experiment had less than 100% microwell occupancy,i.e. some microwells remained empty. The peptide was photoreleased fromthe bead array by 15 min exposure to 365 nm UV source followed byincubation inside a sealed humidified container for 2 hrs. A layer ofCHCA matrix was uniformly deposited across the entire surface of themicrowell plate using the previously described methods. A 6 mm×6 mmsquare region of the microwell plate comprising 13×13 individualmicrowells was imaged by MALDI TOF MSI using the 30 μm raster distancein both X and Y directions, in the positive reflector mode, in the200-2,000 m/z mass range. The acquired MS data was then visualized inseveral mass channels.

FIG. 25A shows MS image of the 6 mm×6 mm region within the microwellplate, which was generated in the 379.5 m/z mass channel. The peak near379 Da is characteristic of the CHCA matrix, therefore the generatedimage shows distribution of the CHCA matrix on the microwell arrayplate. Strong matrix signal is recorded from within the individualmicrowells and also from the top surface of the microwell plate betweenopenings into the microwells. An array of 169 microwells is clearlyvisible in the image in FIG. 25A, however the empty microwells cannot bedistinguished from the bead-occupied microwells in this mass channel.

FIG. 25B shows MS image of the same region within the microwell plate,which was generated in the 1,770-1,775 m/z mass range. This mass rangeencompasses an isotope envelope due to the TRNYYVRAVLGGGSGGSG peptide(SEQ ID NO: 13) (monoisotopic mass 1770.9 Da), which was released fromthe beads after photolysis of the photolabile linker. Because thefabricated array contains only one peptide sequence, the image in FIG.25B shows position of all spots on the microwell array plate thatcontain the peptide analyte TRNYYVRAVLGGGSGGSG (SEQ ID NO: 13)previously bound to the beads. The image in FIG. 25B clearly shows anarray of 169 microwells, some of the microwells being empty and somemicrowells containing spots of the peptide analyte, which are visible aswhite specks localized within a border of a microwell. Limited migrationof the eluted peptide analyte on the surface of the microwell plate alsooccurred in this experiment, which is visible as larger spots thatspread over an area encompassing several microwells.

FIG. 25C shows MS image of the same region within the microwell plate,which was generated in the 228.8 m/z mass channel. A peak near 228 Da isusually assigned to the [M+K]+ ion adduct of the CHCA matrix. Incontrast to the images shown in FIGS. 25A-25B, imaging in the 228.8 m/zmass channel reveals an outer edge of individual microwells, while theinner core of the microwells and the top surface of the microwell plateare not visualized. Imaging in the mass channels adjacent to the 228.8m/z channel or adjusting the intensity threshold of the image in the228.8 m/z channel can additionally visualize the core of the microwells,but not the top surface of the microwell plate. It is noted that onlythe empty microwells, i.e. those that are not occupied by beads weredetected in this mass channel. For example, by comparing the imagesshown in FIGS. 25B and 25C, it can be seen that the peptideanalyte-containing microwells in FIG. 25B generate either very weak orundetectable signal in FIG. 25C. It is likely that the potassium ion,which is likely detected by MALDI TOF MS in the 228.8 m/z mass channel,is desorbed directly from the inner surface of a microwell and thiseffect is greatly reduced when the microwell is occupied by a bead orcontains a peptide.

FIG. 25D shows MS image of the same region within the microwell plate,which was generated in the 1,183-1,206 m/z mass range. This mass rangeencompasses a spectral region that does not contain peaks due to theCHCA matrix or due to the peptide, so only the background intensitypeaks, i.e. the spectral noise should be detectable. Accordingly, theintensity threshold for visualizing the image generated in the1,183-1,206 m/z mass range was set sufficiently low, at 5 a. i. units.Interestingly, the image in FIG. 25D shows a pattern of localized spotsthroughout the measured area, in which the intensity was even lower,below the selected threshold, i.e. no signal at all was detected inthese locations. These spots were localized exclusively withinindividual microwells and most likely reflect the actual position ofindividual microbeads inside a microwell plate. Hydrogel is an extremelypoor substrate for MALDI TOF MS, therefore it is understandable that themass spectra acquired directly from the surface of TentaGel™ beadsexhibit very weak signal. Examination of the array of spots in FIG. 25Dreveals positions of individual beads within their correspondingmicrowells; in fact it can be seen that the beads adhere to thesidewalls of the microwells, in agreement with a microphotograph imageshown in FIG. 22. Furthermore, a diameter of an individual bead wasestimated to be approximately ½ of the diameter of a microwell, which isconsistent with the 90 μm diameter TentaGel™ beads positioned inside the200 μm diameter microwells. Comparison of the images in FIGS. 25B and25D shows that the bead spots and the peptide analyte spots are detectedin the same microwells but appear in different regions within amicrowell. Comparison of the images in FIGS. 25C and 25D furtherconfirms that the microwells containing beads are not visible in the228.8 m/z mass channel. Comparison of the images in FIGS. 25A and 25Dshows that even the very strong 379 Da peak due to the CHCA matrix isnot detected in the areas matching the position of microbeads.

Example 9

Recovery of a Microbead from a Bead Microarray

Microbeads conjugated to a TRNYYVRAVL peptide (SEQ ID NO: 9) via aphotolabile linker were arrayed on an APEX™ glass microwell plate asdescribed previously. Diameter of the beads and dimensions of themicrowells were selected to allow positioning of either one microbeadper microwell or several microbeads per microwell.

The peptide analyte was photoreleased from the bead array and mixed withthe CHCA MALDI matrix using methods described in Examples 6 and 7. Thereleased peptide was subsequently detected by MALDI TOF MS performed ina high lateral resolution mode with 100 single shot spectra collectedfrom each position. The spectra exhibited strong analyte peak atpredicted m/z with the signal-to-noise ratio of 1000:1 as determined bythe instrument software. Following the MS data acquisition, themicrowell array plate was gently rinsed under a stream of deionized H₂Oto remove the residual crystals of CHCA matrix. Throughout thesemanipulations, the microwell plate was inspected under a microscope toverify that the individual microbeads remained inside theircorresponding microwells.

The rinsed microwell plate was air-dried and its surface subsequentlycontacted with 100 μL of 190-proof ethanol applied to an area of theplate that contained the microbeads. Upon evaporation of the solvent,the beads were found outside the microwells on a top surface of themicrowell plate between openings into the microwells. Many of the beadswere found less than 1 mm from their corresponding microwells.Individual beads were removed from the microwell plate and placed intoseparate 1.5 mL microcentrifuge tubes for further analysis.

FIGS. 26A and 26B show a microphotograph of several 90 μm beadspositioned inside a 200 μm wide/200 μm deep microwell (FIG. 26A) and thebeads removed from the microwell by application of ethanol to themicrowell plate as described above (FIG. 26B). The two microphotographswere acquired at a different focus distance.

Example 10

Measurement of an Array of Microbeads by Imaging Mass Spectrometry

A library of microbeads comprising 10 distinct peptide sequences suchthat a single bead was conjugated to a single peptide sequence wasarrayed on a glass microwell plate as described previously. Themolecular weight of individual peptides after their photorelease fromthe beads was calculated to be in the mass range between 1,200 and 1,900Da. Solid phase microparticles of CHCA matrix were deposited insideindividual microwells on top of the beads already placed inside themicrowells as described previously. The peptides were photoreleased andsubsequently mixed with the CHCA matrix inside the microwells asdescribed previously.

The microwell plate was placed on the Opti-TOF plate adapter availablefrom AB Sciex™ and its position secured using Tough-Tags® polyesterlabels. The microwell plate was subsequently loaded into AB Sciex™ 4800MALDI TOF/TOF Analyzer™.

A 6 mm×6 mm square area containing 169 individual microwells was definedby manually selecting the area boundary using the built-in video cameraand entering the selected coordinates into the 4800 Series Imagingsoftware module.

The raster distance was set to 30 μm in both X and Y directions. Themass spectra were acquired from a square grid comprising a total of46656 pixels. The data acquisition was completed within 9 hrs. The imagefile size was approximately 6 GB.

The MS data acquisition parameters were selected as follows: positivereflector mode using the manufacturer-provided instrument settings;500-2,000 m/z mass range; 100 single shot spectra averaged per finalspectrum; laser intensity 4500 relative units. Single shot spectra werecollected in the stationary data acquisition mode. Prior to themeasurement, the instrument was calibrated using the standard MWcalibration mix.

Example 11

Visualization and Analysis of the Microarray Mass Spectrometric ImageData

The microarray image file acquired as described in the previous Examplewas stored in the Analyze 7.5 format by Mayo Clinic (Rochester Minn.)and analyzed using the program BioMap provided by Novartis Institutesfor BioMedical Research.

Spatial distribution of a specific analyte on the microarray wasvisualized in BioMap by selecting an appropriate mass channel matchingm/z position of the analyte monoisotopic peak and then selecting anintensity threshold that was approximately 3-fold greater than thebackground spectral noise. Additional spectral processing algorithmssuch as baseline correction, peak de-isotoping, peak binning and signalnormalization were not utilized in this experiment. In some cases a massrange, which is a combination of several adjacent mass channels, wasused in order to minimize the effect of small variations in a measuredposition of the analyte peak across the microarray. Since the massspectra were recorded from a plain glass surface (not coated with aconductive surface layer), the measured positions of analyte peaks wereexpected to deviate from the predicted values by about 0.5 m/z or lessdue to the static charge accumulation effect.

FIGS. 27A-27D present some of the experimental results obtained usingthe described method. FIG. 27A shows distribution of an analyte measuredin the 568.5 m/z mass channel. The 568.5 Da peak is a characteristicpeak commonly observed in MALDI TOF mass spectra, which is indicative ofCHCA matrix. Therefore the image in FIG. 27A shows distribution of theCHCA matrix on the microarray. The observed pattern matches parametersof the grid of microwells on the microwell plate and confirms that thepresence of CHCA matrix is restricted to the individual microwells. FIG.27B shows distribution of an analyte measured in the 1421.5 m/z masschannel, which is specific for the peptide PPGFSPFRGGGSGGSG (SEQ ID NO:14) released from the bead array after photolysis of the photolabilelinker. Similarly, FIGS. 27C and 27D show distribution of analytesmeasured in the 1771.8 and 1777.0 m/z mass channels, which in thisexperiment were specific for the peptides TRNYYVRAVLGGGSGGSG (SEQ ID NO:13) and ISRPPGFSPFRGGGSGGSG (SEQ ID NO: 15), respectively. Each of themicroarray images in FIGS. 27B-27D shows several spots that exhibit ananalyte signal above the intensity threshold. Several weaker spots alsoappear in each of the FIGS. 27B-27D, which are due to the non-zerointensity background signal measured at the selected m/z position. Thegenerated images of peptide distribution on a microwell plate can beused to identify locations of their corresponding carrier beads withinthe microarray. Analysis of the analyte distribution shows that thepeptides are localized to a single microwell following their releasefrom the beads.

Example 12

Repositioning of an Analyte Released from a Bead Array on a MicrowellPlate

Microbeads conjugated via a photolabile linker to an FITC-labeledpeptide were arrayed on a hydrophobic polystyrene microwell plate usingthe previously described methods. The peptide was partiallyphotoreleased from the beads by a brief 5 minute exposure to 365 nmlight and the UV-irradiated bead microarray was subsequently coated withCHCA matrix applied to the microwell plate using an airbrush methoddescribed in Example 7. The amount of deposited MALDI matrix wassufficient to completely cover the surface of the microwell plate. Thepeptide eluted from its corresponding carrier microbead using thedescribed approach was therefore localized to an immediate vicinity ofthe bead, most likely an outer surface of the bead.

The microwell plate was subsequently placed inside a sealed 50 mLplastic tube containing approximately 2 mL of 50% acetonitrile, 0.2% TFA(v/v) solution. The microwell plate was exposed to the acetonitrilesolution via the vapor phase at room temperature or alternatively at 37°C. Condensation of droplets of liquid on the surface of the microwellplate occurred within several hours in the amount sufficient to dissolvethe original layer of MALDI matrix containing the eluted fluorescentpeptide analyte. Subsequent removal of the microwell plate from thesealed container resulted in rapid evaporation of the solvent andre-crystallization of the peptide-matrix mixture in the areas, whichwere defined by the hydrophobic surface properties of the microwellplate.

FIG. 28 is a series of images showing localization of the fluorescentpeptide analyte, which was first photoreleased from a bead locatedinside a microwell and subsequently repositioned on the microwell plateusing the method described above. The images were acquired at varyingfocus distance on a fluorescence microscope (Olympus Series BHC or LeicaDM4000 B LED). It can be seen that the peptide presence is no longerlimited to an immediate vicinity of its carrier microbeads but extendsto the top surface of the microwell plate between openings into themicrowells (top left image) and also to the sidewalls of individualmicrowells (bottom right image). Furthermore, clusters of CHCA matrixmixed with the fluorescent analyte, which were formed on the microwellplate after the matrix re-crystallization, appear to have uniform size.

Example 13

Localization of a Peptide Analyte-Nanoparticle Mixture on a MicrowellPlate

An array of microbeads conjugated via a photolabile linker to anFITC-labeled peptide was fabricated on a hydrophobic polystyrenemicrowell plate as described in the previous Example. The peptiderelease from the bead array was achieved by contacting the array with anaqueous suspension of silicon dioxide nanoparticles (Sigma-Aldrichcatalog number 56796) additionally containing thermolysin at theconcentration of 50 μg/mL. The reaction between the digestive enzyme andthe peptide substrate was allowed to proceed for 2 hours at roomtemperature within a sealed humidified container. The microwell platewas subsequently removed from the container and air-dried.

FIG. 29 is a series of images acquired on a fluorescence microscope thatshows localization of the fluorescent marker, which was released fromthe bead array by incubation with a digestive compound and subsequentlypositioned on the surface of the microwell plate in the areas adjacentto the openings into the microwells as a mixture with SiO₂nanoparticles. The described method may be useful for analysis of beadarrays by nanoparticle-assisted mass spectrometry.

Example 14

Fluorescence Imaging of a Bead Microarray at Different Focus Distance

The FITC-labeled peptide was partially photoreleased from a bead arrayon the APEX™ glass microwell plate and subsequently mixed with CHCAMALDI matrix, which was achieved by manually dispensing approximately 1μL volume of the matrix solution on the surface of the microwell plateeither in locations adjacent to individual microwells or directly intothe microwells, followed by air-drying.

FIG. 30 is a series of images of the peptide-matrix mixture acquired ona fluorescence microscope using varying focus distance that demonstratesthe ability to separately image individual microbeads located inside themicrowells as well as the eluted peptide analyte located on the bottomsurface of microwells and on the top surface of the microwell platebetween openings into the microwells. Similar approach performed in anautomated mode, e.g. confocal fluorescence imaging may be useful for theseparate imaging of fluorescent analytes that are released from the beadarray versus fluorescent analytes that remain bound to the beads.Furthermore, this approach may be useful for the quantitative assessmentof the extent of the analyte release from the bead array.

Importantly, dispensing only a limited volume of the matrix-containingsolution onto the bead array on a microwell plate using the describedapproach allows elution of a sufficient amount of analyte for detectionand analysis by MALDI mass spectrometry performed from the top surfaceof the microwell plate, but also enables analysis of the individualbeads located inside the microwells by optical methods, i.e. the amountof MALDI matrix deposited inside the microwells is sufficiently small toavoid covering the beads completely.

Example 15

High Resolution Imaging of a Microwell Array Plate by Mass Spectrometry

A surface of a microwell array plate was coated with a thin layer ofCHCA MALDI matrix produced by a nebulizer (PARI Sprint Trek, MidlothianVa.), which is capable of generating microdroplets approximately 3micron in diameter. The matrix-coated microwell plate was subsequentlyimaged on a AB Sciex™ 4800 MALDI TOF-TOF analyzer using the followingparameters: 100 single shot spectra were accumulated in each position,the raster distance was set to 100 micron in both X and Y directions.

FIG. 31 is a microphotograph showing depletion of the CHCA matrix layeron the surface of the microwell plate, which results from the laser beamof the mass spectrometer striking the sample. Four arrows point at asingle spot on the surface, in which the matrix layer has been depletedafter 100 laser shots. Visual examination of the depleted matrix spotindicates that its diameter is approximately 50 micron. Therefore, thespatial resolution of MS imaging provided by the 4800 MALDI TOF-TOFanalyzer in this Example is approximately 50 micron.

Example 16

Fabrication of a Microarray Featuring Multiple Beads Per Microwell

An aqueous suspension of microbeads conjugated to an FITC-labeledpeptide substrate was contacted with a microwell array plate containingmicrowells that are 200 micron deep and 200 micron wide. The beadconcentration was sufficient to completely cover the surface of themicrowell plate. The beads were positioned inside the microwells bycentrifugation at 2,500 RPM and the surplus beads were removed by gentlyrinsing the plate surface with deionized water. FIG. 32 is amicrophotograph of a section of the microwell array plate containingmultiple beads inside each microwell, specifically between 3 and 5 beadsper individual microwell.

Example 17

Reproducibility of the Whole Cell MALDI TOF Mass Spectra

The human breast cancer cell line MCF-7 cells were grown in Gibco® DMEMmedium on a 6-well clear polystyrene cell culture plate (BD Falcon). Thecells were cultured according to the standard molecular biologyprotocols to approximately 50% confluency. The cells were harvested byenzymatic dissociation from the plate using trypsin, re-suspended in1×PBS buffer and stored on wet ice prior to analysis. The cellconcentration was measured to be approximately 1,000 cells per μL.

The MALDI matrix solutions were prepared as follows: (1) 10 mg/mL ofalpha-Cyano-4-hydroxycinnamic acid (CHCA) dissolved in 50% acetonitrileand 0.2% TFA; and (2) 10 mg/mL of sinapinic acid (SA) dissolved in 50%acetonitrile and 0.2% TFA. Approximately 20 μL of cell suspension in PBSbuffer was thoroughly mixed with an equal volume of MALDI matrixsolution and let stand in a capped plastic microcentrifuge tube for 30min at room temperature.

The cell-MALDI matrix mixture was then re-suspended by pipetting andspotted in ten adjacent spots on a stainless steel Opti-TOF 384-wellMALDI target plate using 1 μL of mixture per spot. The diameter of eachspot was approximately 2 mm.

The MALDI TOF MS data was acquired on the AB Sciex™ 4800 MALDI TOF/TOFAnalyzer using factory-default instrument settings in the positivelinear mid-mass mode, in the mass range between 2,000 and 25,000 m/z,laser power of 4500 relative units, averaged 4,000 single shot spectraper spot. The data was collected using the random spectral acquisitionpattern provided by the 4000 Series Explorer™ software.

FIG. 33 shows representative whole cell mass spectra acquired using SAmatrix after collecting 4,000 laser shots from a sample spot. The threespectra represent mass spectra independently acquired from threedifferent sample spots. The spectra are shown in the 7,500-9,100 m/zmass range, a subsection of the entire 2,500-25,000 m/z range. Theintensity scale (Y axis) is in absolute intensity (a.i.) units. Overall,the spectral data is highly reproducible and even peaks with very lowintensity (peak that have less than 1% relative intensity) arereproducibly detected in every spectrum. Furthermore, the spectra arehighly informative: over 100 unique spectral features were identifiedjust in the shown region and over 1,000 peaks were detected in theentire 2,000-25,000 m/z mass range.

Example 18

Difference Mass Spectrometry

Reproducibility of the whole cell mass spectra was further demonstratedusing the method of difference mass spectrometry disclosed in thisspecification. Two mass spectra independently acquired as described inthe previous Example were loaded into mMass open source massspectrometry tool and baseline-corrected using the provided mathematicalalgorithm. The spectra were normalized, which was achieved bymultiplying each spectrum by an experimentally determined coefficient,such that the intensity of the most prominent peak near 5,000 m/z wasapproximately equal in both spectra.

The spectral subtraction procedure was carried out using the spectralsubtract option available with mMass open source mass spectrometry tool.The two original spectra and the resulting difference spectrum are shownin FIG. 34 as top, middle and bottom traces labeled “sample 1”, “sample2” and “sample 1-sample 2”, respectively. The spectral subtract datafurther confirms that mass spectra independently acquired from identicalcells are highly reproducible, i.e. the position and relative intensityof individual peaks are nearly identical in such spectra. Smalldifferences, which are observed as weak peaks with either positive ornegative intensity in the resulting difference spectrum, can beexplained in part by imprecise digitization of a peak position, theeffect known as spectral jitter. It is expected that the latestgeneration MALDI TOF mass spectrometers and FT-MS instruments will becapable of producing even more accurate spectral data.

Example 19

Difference Mass Spectrometry Applied for Detection of Spectral ChangesDue to Apoptosis

The human breast cancer cell line MCF-7 cells were grown in Gibco® DMEMmedium on a 6-well clear polystyrene cell culture plate (BD Falcon). Thecells were grown according to the manufacturer's protocol toapproximately 75% confluency. At this point several apoptosis-inducingcompounds were added to the individual wells and the cells were furtherincubated for 24 hrs. Both live adherent cells and dead cells detachedfrom the surface were collected from the individual wells of the cellculture plate and the cell viability count was performed on Countless®automated cell counter (Life Technologies) per manufacturer's protocol.

The cells for WCMS analysis were prepared as described in the previousexamples. The cell concentration was approximately 1,000 cells per μL.

Whole cell MALDI TOF MS spectra were acquired from the control(untreated) and treated cell populations using instrument parametersdescribed in the previous examples. FIG. 35 shows representative WCMSspectra acquired from the untreated (top spectrum) and theEtoposide-treated (middle spectrum) cell populations. The bottomspectrum represents a difference mass spectrum produced by subtractingthe control spectrum from the Etoposide-treated spectrum. Note thatdespite the overall similarity of the top two spectra, the differencemass spectrum reveals multiple spectral features, some of which may beassociated with the apoptosis-induced changes in the cell composition.

Example 20

Culture of Adherent Cells on Microbeads Analyzable by WCMS

Glass microcarrier beads (Sigma-Aldrich catalog number G2767, individualparticle size 150 to 210 μm) were sterilized by autoclaving and added toindividual wells of a 6-well polystyrene cell culture plate. The humanbreast cancer cell line MCF-7 cells were grown in Gibco® DMEM medium asdescribed in the previous Example. Upon examination under a microscope,growth of adherent MCF-7 cells was observed on the surface of individualbeads as well as on the inner surface of the wells. The beads wererecovered from the wells, rinsed in 1×PBS buffer and deposited on theOpti-TOF 384-spot MALDI target plate at one bead per spot. The beadswere overlaid with 0.5 μL of SA matrix dissolved in 50%acetonitrile/0.2% TFA and air-dried. MALDI TOF mass spectra acquiredfrom the individual spots on the Opti-TOF plate are thereforerepresentative of a cell population bound to a single microbead.

FIG. 36 shows WCMS spectra of the MCF-7 cells independently acquiredfrom three different glass carrier microbeads. The spectra are highlysimilar indicating that not only the spectral acquisition process isreproducible as described in the Example 17, but also the cell growthconditions are highly reproducible for the individual beads, whichresults in the identical cell composition.

In a separate experiment, the MCF-7 cells grown on beads were treatedwith apoptosis inducing compounds. The WCMS spectra acquired from thecontrol (untreated) and the treated beads were considerably differentindicating that changes in the cellular composition due to apoptosis canbe detected in the spectra acquired from a single microbead.

Example 21

Whole Cell Mass Spectra Acquired from an Array of Microbeads on aMicrowell Plate

The MCF-7 cell line cells were grown attached to the glass microcarrierbeads as described in the previous Example. Individual microbeads withbound cells were subsequently removed from the cell culture medium andexamined under a microscope to determine the cell density, the totalnumber of cells attached to a single bead and in some cases to evaluatethe cell morphology. FIG. 37A is a representative microphotograph of amicrobead with multiple cells bound to the bead surface. The totalnumber of cells bound to a single bead was estimated to be greater than100. As shown below, that amount was sufficient for acquiring highlyinformative whole cell mass spectra from a single bead.

An aliquot containing several hundred microbeads with attached cells wasrinsed in PBS buffer and distributed into individual wells of amicrowell array plate using the previously described methods. Throughoutthis procedure the cells remained bound to the bead surface. FIG. 37B isa representative microphotograph of a section of a microwell array platewith some empty microwells and some microwells containing a singlemicrobead with multiple bound cells. Note that in this Example thediameter and depth of microwells were selected to be larger than thediameter of microbeads in order to also accommodate the cells bound tothe bead surface.

The array of cell-conjugated microbeads fabricated on a microwell plateas depicted in FIG. 37B was subsequently coated with a solutioncontaining sinapinic acid at the concentration of 10 mg/mL and subjectedto MALDI TOF MS analysis performed in a high spatial resolution mode.Specifically, the mass spectra were acquired from a square grid of spots(pixels) separated by 50 μm raster distance in both X and Y orthogonaldirections. Exemplary whole cell mass spectra acquired from the cellsbound to microbeads arrayed on a microwell plate using the describedapproach are shown in FIG. 37C. The two mass spectra shown in FIG. 37Cwere acquired independently from different regions within a microwellplate, i.e. from different microbeads and represent averaged datacollected from four adjacent pixels within a 100 μm×100 μm area, thedata from each pixel comprising 50 averaged single-shot spectra. Thespectra acquired from different microbeads were highly reproducibleincluding both the position and the intensity of individual peaks.

In a separate experiment, the MCF-7 cell line cells were grown on beadsas described above and labeled with a fluorescent marker while stillattached to the beads using the PKH67 Green Fluorescent Cell Linker MidiKit for general cell membrane labeling (Sigma-Aldrich, St. Louis Mo.)following the manufacturer's protocol. Labeled cells bound to the glassmicrocarrier beads were arrayed on a microwell plate and examined usinga fluorescent microscope. In this experiment spatial distribution of thefluorescent label was also measured after the microwell plate was coatedwith the MALDI matrix but prior to the WCMS analysis in order to measurethe extent of cell rupture that occurs upon the cell contact with theMALDI matrix solution and subsequent crystallization of the matrix. Bothlateral migration of the fluorescent compound on the microwell platesurface and its depth profile within individual microwells weremeasured.

Example 22

Cell Culture and Optical Analysis Performed on a Microwell Array Plate

The MCF-7 cell line cells were grown on an APEX™ glass microwell arrayplate or alternatively on a fiber optic glass microwell plate using astandard cell culture protocol. The morphology of individual cells andtheir localization on the plate surface were examined using an opticalmicroscope. FIG. 38 is a series of images of a representative region ofan APEX™ glass microwell plate containing a single microwell. Theindividual images were acquired at different focus distance of theoptical microscope. The images in FIG. 38 show individual MCF-7 cellsattached to the top surface of the plate between openings into themicrowells, to the sidewalls of the microwells and to the bottom surfaceof the microwells. The cells found in these topologically distinctregions of the microwell plate exhibited similar or identical morphologyindicating that the growth conditions were similar or identical insideand outside the microwells. Furthermore, the morphology of the cellscultured on the APEX™ glass and fiber optic plates, as measured byoptical imaging, was found to be very similar to the control MCF-7 cellscultured under identical conditions using either the industry-standardflat microscope slides coated with polylysine or a Petri dish. It isnoted that the cells readily grow on the unmodified surface APEX™ glassplates; for example in many experiments the amount of time needed toreach a specific cell density on those plates, e.g. 50% confluency wasnearly identical to the amount of time needed to reach the same celldensity on a standard polylysine-coated microscope slide or on a Petridish. It is concluded that the examined glass microwell plates can serveas suitable solid supports in a variety of cell culture applicationsincluding the drug screening applications as described in detailelsewhere in this specification. Furthermore, adherent mammalian cellscultured on the glass microwell plates examined here and on theconventional solid supports, such as a Petri dish appear to be similarphysiologically, which is supported by the experimental optical and massspec data presented in the instant specification.

In a separate experiment, the cells were cultured as described above onmicrowell plates featuring microwells of different diameter and thetotal number of cells localized inside individual microwells wascounted. Microwells as small as 100 μm in diameter contained close to100 individual MCF-7 cells, an amount sufficient for high-sensitivitydownstream analysis by WCMS. Furthermore, it was experimentally verifiedthat the quantity of individual cells localized within adjacentmicrowells on a microwell plate was similar, e.g. did not differ by morethan 25%, In some cases the quantity of cells localized insideneighboring microwells did not differ by more than 10%.

In a separate experiment, the cells cultured on a microwell array plateas described above were stained using PKH67 Green Fluorescent CellLinker Midi Kit for general cell membrane labeling (Sigma-Aldrich, St.Louis Mo.) following the manufacturer's protocol and the distribution ofcells on the plate was visualized using a fluorescence microscope.

Example 23

Cell Culture and WCMS Analysis Performed on a Microwell Array Plate

A multi-chamber cell culture device was assembled as schematicallydepicted in FIG. 15A by affixing a sterile multi-well silicone gasket toa top surface of a sterile microwell array plate. The multi-well gasketcomprised either 12 or 16 square wells arranged in two rows, each wellmeasuring approximately 7.5×7.5×8 mm as width×length×height. Themicrowell array plate was manufactured from the APEX™ glass oralternatively from a fused fiber optic bundle. The cell culture devicefurther included a plastic lid positioned on top of the silicone gasket.The adherent MCF-7 cell line cells were seeded into individual wells ofthe multi-well gasket and grown on the plate using a standard cellculture protocol. After 48 hours of incubation the gasket was removed asschematically depicted in FIG. 15C and the surface of the plate withattached cells was gently rinsed with PBS buffer. Alternatively, thegasket was separated from the microwell plate after the PBS rinse step.The surface of the plate containing bound cells was immediately coatedwith a layer of SA MALDI matrix using the previously described airbrushmethod.

WCMS analysis was performed on AB Sciex 4800 MALDI TOF/TOF Analyzer inthe linear positive mode in the 2,500-25,000 m/z mass range using thefactory-default mid-mass positive data acquisition and interpretationmethods. The data acquisition was performed in the high spatialresolution mode, i.e. each mass spectrum was acquired within an areadefined by the diameter of the instrument ionization laser beam, whichwas approximately 50 μm. Accordingly, over 20,000 mass spectra werecollected from an area of the plate corresponding to a single well of amulti-well gasket.

In a separate experiment, the MCF-7 cells were cultured using amulti-chamber cell culture device fabricated from a multi-well siliconegasket affixed to: (1) a flat surface microscope slide that did notcontain microwells; (2) a microwell plate featuring 100 μm deepmicrowells; (3) a microwell plate featuring 200 μm deep microwells. Themicroscope slide and the microwell plates with the surface-bound cellswere subjected to WCMS analysis as described previously. Overall qualityof the mass spectral data and the signal intensity were assessed foreach of the three described configurations.

Example 24

Fabrication of a Live Cell Microarray on a Microwell Plate

The live cell microarray was fabricated as follows: a sterile 12-wellsilicone gasket was affixed to a top surface of a sterile APEX™ glassmicrowell plate, as schematically depicted in FIG. 15A and 1×DMEM cellculture medium supplemented with 10% FBS and 1% ofPenicillin/Streptomycin was dispensed into each well of the fabricated12-chamber cell culture device. Several hundred of MCF-7 cell line cellswere seeded into each of the 12 wells and the cells were grown to ˜80%density on the microwell plate within 48 hrs using a standard cellculture protocol. The fabricated live cell microarray thus comprised 12reactive sites defined by the dimensions of the silicone gasket, eachreactive site measuring approximately 7.5 mm×7.5 mm and containingapproximately 5×10⁵ live cells. Visual inspection of the fabricatedmicroarray was performed to verify that the cells were bound to the topsurface of the microwell plate between openings into the microwells, tothe sidewalls of individual microwells and to the bottom surface ofindividual microwells.

In this example all reactive sites of the fabricated live cellmicroarray contained identical cells, which were grown using identicalconditions. However, the described method can be easily modified suchthat the individual reactive sites of the microarray will containdifferent cell types. Alternatively, the individual reactive sites ofthe microarray may contain identical cells, which were grown underdifferent conditions, for example in a cell growth medium supplementedwith different chemical compounds. This is possible because theindividual reactive sites of the microarray, i.e. the clusters of cellsare separated from each other on the surface of the microwell plate bythe walls of the silicone gasket, thereby eliminating the risk of cellcross-contamination.

It also should be understood that although in this Example a microwellplate was utilized as a solid support for fabricating a cell microarray,conventional flat-surface slides may be utilized in certain instances,as long as the slide surface is compatible with analysis by massspectrometry.

Example 25

Reacting the Live Cell Microarray with Drug Compounds

The live cell microarray fabricated as described in the previous Examplewas utilized to measure response of the MCF-7 cell line cells to severalapoptosis-inducing compounds, which included etoposide, camptothecin andandrographolide. In addition, the live cell microarray was utilized tomeasure response of the MCF-7 cells to the same compound(andrographolide) provided in three different concentrations.

Stock solutions of the three compounds listed above were prepared inDMSO and stored at 4° C. or at −20° C. The compounds were diluted withthe DMEM cell culture medium to the final concentrations of 1 mM(etoposide), 10 μM (camptothecin) and 100 μM, 500 μM and 1 mM(andrographolide) immediately before use. The original cell growthmedium within the individual wells of the silicone gasket was removed bypipetting and replaced with approximately 250 μL of a cell growth mediumsupplemented with a specific compound. FIG. 39A schematically depicts alayout of the cell microarray. CT denotes control spots, i.e. cellsexposed to the drug-free cell growth medium, E denotes etoposide spots,C denotes camptothecin spots, A1, A2 and A3 denote andrographolide spotsin which the drug concentration was 100 μM, 500 μM and 1 mM,respectively. Each of the compounds was tested in duplicate. BL denotesblank spots that contained no cells.

Cells within the cell microarray were continuously treated with the drugcompounds for 24 hours. The reaction was subsequently stopped; themicroarray was rinsed with 1×PBS buffer and immediately coated with alayer of sinapinic acid MALDI matrix using the airbrush method of matrixdeposition. The multi-well silicone gasket was detached from themicrowell plate either prior to or following the PBS rinse step.

The reacted cell microarray was measured by MALDI TOF MS in the linearmid-mass positive mode as described previously. Individual spectracontained 50 averaged single-shot spectra and were acquired from spots,which were spaced apart by 100 μm in two orthogonal directions.

Example 26

Analysis of the Cell Microarray Image Data

FIG. 39B is an MS image of the reacted cell microarray, which wasvisualized in the 2,000-25,000 m/z mass range using the “maximum signal”option provided by the BioMap analytical software. Twelve distinctregions arranged in two rows can be seen in the image. The shape anddimensions of the individual regions match the footprint of themulti-well silicone gasket, which was used to fabricate the microarray.The rightmost section of the image in FIG. 39B contains two blank spots(top and bottom), which were not covered by the multi-well gasket and inwhich no cells were present.

FIG. 39C is an MS image of the top row of spots within the reactedmicroarray, which was visualized in the 10,431 m/z mass channel. Astrong peak near 10,431 m/z was detected in the mass spectra acquiredfrom every reactive site of the microarray including both the controlcells and the cells treated with etoposide, camptothecin orandrographolide. On the other hand, no peak at this position wasdetected in the mass spectra acquired from the blank spots. Theexperimental data confirms that the three-dimensional structure of amicrowell array plate efficiently retains individual cells in thespatially distinct regions of the microwell plate even if the cells areonly loosely bound to the surface of the plate or detach from thesurface.

FIG. 39D is an MS image of the top row of spots within the reactedmicroarray, which was visualized in the 8,556 m/z mass channel. A peaknear this position can be tentatively assigned to ubiquitin, which has amolecular weight of 8,564 Da. Although the 8,556 m/z peak was present inall of the measured mass spectra, its intensity was significantlygreater in the spectra of untreated cells. Therefore, this spectralfeature has been used to distinguish the untreated cells from the cells,which were treated with any of the above compounds.

FIG. 39E is an MS image of the top row of spots within the reactedmicroarray, which was visualized in the 7,891-7,977 m/z mass channel.Several peaks are present in the 7,891-7,977 m/z spectral region thathave significantly greater intensity in the spectra of the cells treatedwith etoposide, camptothecin or andrographolide compared to the spectraof untreated cells.

FIG. 40A shows representative whole cell mass spectra, which wereacquired from the blank spots (labeled BL), untreated control cells(labeled CT), cells treated with camptothecin (labeled C), etoposide(labeled E) and 1 mM andrographolide (labeled A3). FIG. 40B showsrepresentative whole cell mass spectra, which were acquired from thecells treated with 100 μM, 500 μM and 1 mM andrographolide, the spectralabeled A1, A2 and A3, respectively. The mass spectra shown in FIGS.40A-40B comprise 200 averaged single-shot spectra, which were acquiredfrom a 200 μm×200 μm square area located within the individualmicroarray reactive sites. It can be seen that the acquired spectra arehighly informative, i.e. contain multiple spectral features, which maybe utilized in a search for potential biomarkers of a cell condition. Asa non-limiting example, examination of the peaks located in the10,000-15,000 m/z spectral region may be utilized to detect spectraldifferences associated with changes in the post-translationalmodifications of various histone proteins including the type and extentof the post-translational modifications. Although the live cellmicroarray described in this Example contained 12 reactive sites, it isof course possible to accommodate much greater number of reactive siteson a single 25×75 mm microchip. In fact, microarrays containing over athousand reactive sites on a single microchip may be fabricated usingthe described methods. The smaller individual reactive sites willnevertheless contain sufficient amount of analyte for analysis by massspectrometry and optionally also by optical imaging.

Example 27

Composite Microbead-Microwell Live Cell Microarray

Several cation-exchange and anion-exchange resins were purchased fromSigma-Aldrich (St. Louis Mo.), including CM Sephadex® C-50,DEAE-Sephadex® A-25 chloride form, Dowex® Marathon™ A, chloride form,Diaion® WA30 free base, Dowex® 1×4 chloride form, 20-50 mesh,DEAE-Sepharose® and preswollen microgranular CM-cellulose.

The individual beads were soaked overnight in a saturated solution ofetoposide in DMSO and subsequently arrayed inside 200 μm deep, 200 μmwide microwells on an APEX™ glass microwell array plate using thepreviously described methods. Depending on the bead diameter, either onebead or several beads were placed inside each microwell. Regions of themicrowell plate containing different bead types were separated by amulti-well silicone gasket affixed to the top surface of the microwellplate. For each bead type, a control spot was also provided, whichcontained either beads of the same type rinsed in deionized water oralternatively empty microwells.

The fabricated microbead-microwell microarray thus comprised 14 reactivesites, of which 7 sites contained etoposide bound to an ion-exchangemedium; each reactive site comprised 169 microwells within a 6 mm×6 mmsquare area with individual microwells filled with a particular type ofan ion-exchange resin.

Individual wells of the multi-well silicone gasket were filled with 250μL of the DMEM cell culture medium containing approximately 10,000 ofthe MCF-7 cell line cells. In this Example, the release of etoposidefrom the beads into the cell culture medium occurred primarily viadiffusion mechanism, although other methods, such as photorelease ofactive compounds from individual beads may be also utilized due to thefact that the solid support used for the microarray fabrication issufficiently transparent in the UV-visible range. In a separateexperiment, the etoposide-containing ion exchange medium was addeddirectly into individual wells of the multi-well gasket after the wellswere filled with the DMEM cell culture medium containing the MCF-7cells.

The cells were continuously exposed to the etoposide-containing cellculture medium or alternatively to the drug-free cell culture medium for24 hours. Upon the reaction completion the cell microarray was analyzedusing several methods.

Optical imaging: the multi-well gasket was separated from the microwellarray plate, the surface of the plate rinsed with 1×PBS buffer and themicrowell plate was imaged using an optical microscope to determine thecell density and cell morphology. A representative image acquired froman area of the microwell plate, which contained the cells exposed to thedrug-free DMEM medium, is shown in FIG. 41A. A representative imageacquired from an area of the microwell plate, which contained the cellsexposed to the etoposide-containing DMEM medium, is shown in FIG. 41B. Asignificantly greater density of adherent cells was observed for thecells grown in the drug-free medium.

Cell counting: the adherent cells were separated from the surface of themicrowell plate by enzymatic digest and the ratio of live to dead cellsmeasured using an automated cell counter after the cells were stainedwith Trypan blue. Representative cell count data obtained from thedrug-free cell growth medium: 9.2×10⁵ total cells, 6.1×10⁵ live cells,67% viability. Representative cell count data obtained from the cellgrowth medium supplemented with etoposide released from the ion exchangebeads: 4.7×10⁵ total cells, 2.1×10⁵ live cells, 44% viability.

Mass spectrometry: the multi-well gasket was separated from themicrowell array plate, the surface of the plate rinsed with 1×PBS bufferand immediately coated with the SA MALDI matrix using the previouslydescribed airbrush method of matrix deposition. The microwell arrayplate surface was subsequently measured by MALDI TOF mass spectrometryin the linear positive mode as described previously.

Example 28

A Microwell Array Plate with a Surface Layer of Immobilized DigestiveEnzyme

A microwell array plate comprising an array of cylindrical microwellsmeasuring 200-250 μm diameter, 180±25 μm deep with 500 μm pitch werefabricated from APEX™ photo-structured glass by Trianja Technologies Inc(Allen Tex.) using the acid etch technology and subsequentlysurface-activated with a reactive epoxy layer by the manufacturer. Theepoxy layer was deposited on the inner surface of individual microwells,e.g. sidewalls of the microwells and bottom surface of the microwells inaddition to the space between openings into the microwells.

TPCK treated trypsin from bovine pancreas was purchased fromSigma-Aldrich. The enzyme was reconstituted in 1×PBS buffer at 1 mg/mLconcentration. The enzyme conjugation to the epoxy activated surface ofthe microwell array plate was performed essentially as described in theapplication note for protein conjugation to SuperEpoxy microarraysubstrates published by Arrayit corporation, the website version of theapplication note accessed Feb. 25, 2013. In order to facilitate entry ofthe enzyme-containing solution into the microwells, the microwell platewith the solution was briefly placed into a vacuum chamber, which causedair bubbles to escape from the microwells.

To verify that a surface layer of immobilized enzyme in the active(functional) form was formed on the microwell plate, an aqueous solutionof bovine serum albumin (BSA) at the concentration of 1 mg/mL wasspotted in several locations across the microwell plate and incubatedfor 3 to 4 hrs at 37° C. The occurrence of proteolytic degradation ofBSA by the surface-immobilized trypsin was confirmed via acquisition ofMALDI TOF mass spectra in the selected locations and detection of peaksin the mass spectra that corresponded to known proteolytic fragments ofBSA.

Example 29

In-the-Microwell Digestion of Analytes Released from a Bead Array

The microwell plates containing an immobilized surface layer of trypsinfabricated as described in the previous Example were used to performdigestion of polypeptides eluted from individual beads in a bead arrayformat.

For analytes conjugated to beads via a photolabile linker, the analyteelution was achieved by: (i) making a bead array on thetrypsin-immobilized microwell plate, (ii) removing the solvent and (iii)exposing the dry bead array to the 365 nm UV source for 15 min. The beadarray was subsequently re-hydrated by exposing the microwell plate to anaerosol containing dI H₂O or 100 mM ammonium bicarbonate buffer suchthat droplets, which formed on a surface of the microwell plate, did notmerge between any two adjacent microwells. The bead array was incubatedat 37° C. for at least 2 hours and sometimes as long as overnight insidea humidified container, then coated with a layer of CHCA matrix andmeasured by MALDI TOF mass spectrometry in the imaging mode.

For analytes conjugated to beads via an affinity interaction, e.g.antibody-antigen, the analyte elution was achieved by exposing themicrowell plate to an aerosol containing an acidic solution, such as0.1% TFA, then drying the plate and applying an aerosol containing theammonium bicarbonate buffer.

Efficient enzymatic digestion was observed for analytes that have beeneluted from the beads but was minimal for analytes that remainedconjugated to the beads, e.g. multiple peaks corresponding to variousfragments of the eluted antigen were detected in the mass spectrarecorded off the microwell plate but few, if any, peaks corresponding tofragments of the bead-conjugated antibody, were observed. Theinterpretation of the experimental data is that the sample, e.g. aprotein or a polypeptide needs to be physically separated from itscarrier bead and brought in contact with the surface layer of thedigestive enzyme in order to be fragmented by the enzyme. This effectenables detailed study of the structure of bead-conjugated proteincomplexes in a bead microarray format. It was also observed that theefficiency of enzymatic digestion varied depending on the duration ofthe incubation.

The described technique provides an efficient method for confirmingseparation of an analyte from its carrier bead, which may include thefollowing steps: (i) contacting the analyte-conjugated bead with adigestive enzyme-coated solid support, (ii) providing a sufficientamount of liquid medium, e.g. dI H₂O to enable migration of the analytefrom the bead onto the solid support and (iii) analyzing the solidsupport by mass spectrometry to detect the presence of proteolyticfragment(s)

Example 30

In-the-Microwell Digestion of Analytes from a Bead Array Using aNanoparticle-Encapsulated Digestive Enzyme

TPCK treated trypsin from bovine pancreas was purchased fromSigma-Aldrich. The enzyme was encapsulated into poly(lactic-co-glycolicacid) or PLGA nanoparticles with 500 nm average diameter at 10% loadingpercentage. The copolymer type was 50:50 PLGA 1A, which provides thefastest dissolving time. Some nanoparticles were additionally labeledwith FITC fluorescent dye. The enzyme encapsulation service wasperformed by a commercial entity specializing in custom formulations fornanoparticle-based drug delivery applications.

To determine whether the PLGA-based materials are compatible withanalysis by mass spectrometry, the copolymer was hydrolyzed in dI waterat room temperature for one week and the spectra measured by MALDI TOFMS. Multiple peaks were observed in the mass spectra, consistent withthe presence of various oligomers, however the majority of peaks due toPLGA were below 900 m/z and almost no peaks were detected above 1,200m/z. Therefore the spectral region above 1,200 m/z is free of spectralinterference from the hydrolyzed PLGA and can be used for detection ofproteolytic fragments of polypeptide and protein analytes.

The bead array comprising TENTAGEL® bead-conjugated polypeptides wasfabricated on a microwell array plate and a layer of trypsin-containingnanoparticles was subsequently deposited on top of the bead array bycentrifugation. Both the beads and the nanoparticles were localizedentirely within individual microwells.

The fabricated array of beads and nanoparticles was placed inside ahumidified chamber and incubated for a specific amount of time. Theduration of incubation was 12 hrs, 24 hrs, 2 days, 4 days, 6 days, 12days and 21 days. Proteolysis of the bead-conjugated polypeptides wasinitiated by release of the digestive compound (trypin) from thehydrolyzed nanoparticles, which took place in the aqueous medium. Foreach duration of incubation the bead array was subsequently analyzed bymass spectrometry to determine occurrence and extent of the proteolysisreaction. The bead arrays were also analyzed by fluorescence in the 532nm excitation channel to detect localization of FITC, which is releasedfrom the fluorescent-labeled nanoparticles upon hydrolysis. Peaks in themass spectra indicative of proteolysis were detected as early as 12 hrsafter contacting the bead array with the nanoparticle encapsulateddigestive enzyme. It was estimated that less than 5% of the total enzymewas released into the aqueous liquid medium during the first 1 hour ofincubating the nanoparticles with the aqueous medium, likely less than1%. It was estimated that greater than 10% of the total enzyme wasreleased into the aqueous liquid medium after 12 hours of incubating thenanoparticles with the aqueous medium, likely greater than 25%.

Several conclusions are drawn from this study. First, it is possible andindeed convenient to perform an enzymatic digestion reaction bycontacting a bead array fabricated on a microwell array plate with adigestive compound, which is in the solid state, for example in the formof slowly dissolving microcrystals or in the form of nanoparticles. Thisapproach ensures localization of the digestion reaction withinindividual microwells and provides time-controlled release of thedigestive compound upon contact with a liquid medium and its subsequentreaction with the sample, which may be beneficial for studying thereaction kinetics.

Second, it is noted that the described method involving time-dependentrelease of an active agent from nanoparticles and its subsequentreaction with compounds conjugated to microbeads individually arrayedinside microwells on a microwell plate may be broadly applicable to avariety of studies that utilize nanoparticles. In particular, variousdrug development and drug delivery research studies may utilize thedescribed approach to evaluate reaction between a drug candidate, e.g. asmall molecule and its intended target, e.g. a protein. Hundreds ofdistinct proteins may be individually conjugated to the beads forming aprotein bead array or an antibody bead array for studying specificity ofa nanoparticle-encapsulated compound. In this approach, both thespecificity of the drug-target interaction and the properties ofnanoparticles may be conveniently probed in a microarray format.

Example 31

Peptide Bead Library Comprising Compounds Identifiable by MS-MSSequencing and/or Enzymatic Digestion

A series of bead-conjugated peptides were synthesized using Fmoc solidphase chemistry on 90 μm diameter TENTAGEL® beads (“TG Beads”). Thepeptide sequences were: VFDRGGGSGGSG-PLL-Ahx-TG Bead (SEQ ID NO: 16),VFRDGGGSGGSG-PLL-Ahx-TG Bead (SEQ ID NO: 17), VDFRGGGSGGSG-PLL-Ahx-TGBead (SEQ ID NO: 18), VDRFGGGSGGSG-PLL-Ahx-TG Bead (SEQ ID NO: 19),VRFDGGGSGGSG-PLL-Ahx-TG Bead (SEQ ID NO: 20) and VRDFGGGSGGSG-PLL-Ahx-TGBead (SEQ ID NO: 21) where PLL is a photolabile linker cleavable by 365nm light described previously and Ahx is an aminohexanoic acid spacer.The synthesis scale was 5 μmol. The peptide purity was “on resin”, i.e.the peptides were used without further purification. The peptidecompounds had an identical molecular weight but different amino acidsequence. The peptide compounds therefore had different MALDI TOF-TOF MSfragmentation spectra and different digestion profiles after incubationwith proteinase K, thermolysin, trypsin and pronase, which enabledidentification of the peptide sequences released from the otherwiseidentical beads using mass spectrometry.

Example 32

Peptide Bead Library Comprising Multiple Active Agents Conjugated to aSingle Bead

A series of bead-conjugated peptides were synthesized using Fmoc solidphase chemistry on 90 μm diameter TENTAGEL® beads (“TG Beads”). Thesynthesis scale was 5 μmol. The peptide purity was “on resin”, i.e. thepeptides were used without further purification. The peptide sequenceswere: [5-FAM]VFZDGGGSGGSG-PLL-Ahx-TG Bead (SEQ ID NO: 22) and[5-FAM]VFDZGGGSGGSG-PLL-Ahx-TG Bead (SEQ ID NO: 23), where [5-FAM] is5-carboxyfluorescein, PLL is a photolabile linker cleavable by 365 nmlight, Ahx is an aminohexanoic acid spacer and Z is a mixture of 19native amino acids (excluding Cys) in an approximately equimolar ratio.Therefore, each bead was conjugated to 19 distinct peptide sequences.Because each peptide sequence was present on a bead in sufficient amountand remained accessible to a sample, e.g. an enzyme solution contactingthe bead, the described peptide-bead compositions enable reactionmultiplexing at the single-bead level.

Example 33

Detection of an Enzymatic Reaction on a Bead Microarray by Fluorescenceand Mass Spectrometry

The fluorescent peptide-conjugated 90 μm diameter beads described in theprevious Example were arrayed inside individual ˜220 μm diametermicrowells on a microwell array plate using previously describedmethods. A microphotograph of the microwell plate in FIG. 42A and azoom-in in FIG. 42B reveal positions of individual beads within thewells. The fabricated bead array was subjected to an enzymatic digestionreaction using an aqueous solution of trypsin at 3 mg/mL delivered tothe bead array in the form of an aerosol. The bead array was incubatedwith the enzyme solution for at least 2 hrs inside a humidified chamberat 37° C.

The reacted bead array was subsequently dried and first analyzed byfluorescence imaging on NIKON Widefield inverted TE2000 imaging systemequipped with prior motorized X, Y stage and a HAMAMATSU ORCA ER digitalCCD camera. The acquired fluorescence images were processed and analyzedusing the associated NIKON Elements™ software.

Elution of fluorescent analyte from the individual beads into themicrowells was detected in the acquired image as shown in FIG. 42C. Themicrowell plate was subsequently coated with a layer of CHCA MALDImatrix and measured on a 4800 MALDI TOF mass spectrometer in order toidentify proteolytic fragments released from the individual beads.

In this example, the occurrence of a proteolytic reaction, i.e. theevent of release of fluorescent analyte(s) from the beads was detectedby fluorescence imaging followed by analysis by mass spectrometry todetermine molecular weights of the released analytes in order toidentify the peptide reactive substrates as well as to map the proteaserecognition sites. The mass spectrometry analysis was performed in theimaging mode. In a separate experiment, the acquisition of the mass specdata was limited to individual microwells that contained the beads andwithin such microwells, to portions of the wells that contained theeluted analytes and were not occupied by the beads.

Example 34

Fabrication of a Reference Microwell Plate for Testing Conditions ofAnalyte Elution from a Bead Array

It is sometimes desirable to provide an end-user with a test microwellplate, which could be used for optimizing experimental proceduresrelated to the analyte elution from a bead array and localization of theeluted analyte on a microwell plate. For example, such test microwellplates could be useful for optimizing conditions of the MALDI matrixdeposition using a nebulizer, an airbrush, a spotting robot, a TLCsprayer or a similarly functioning device. The analytes have fluorescentproperties and their distribution on the microwell plate after elutionfrom the beads can be visualized by 2D or 3D fluorescence imaging usinga conventional microarray scanner or a fluorescence microscope.

The bead array was fabricated on an optically clear plastic cyclicolefin copolymer (COC) or a cyclic olefin polymer (COP) microwell arrayplate manufactured by the soft embossing method. Individual microwellswere 250 μm diameter and 250 μm deep, separated by 350 μm measured as adistance between centers of adjacent microwells. The microwells formed asquare grid. The 90 micron TENTAGEL® beads were conjugated to the[5-FAM]VFZDGGGSGGSG-PLL-Ahx peptide sequence (SEQ ID NO: 26), which wasdescribed previously. The amount of fluorescent peptide analyte bound toa single bead was approximately 500 pmol. A suspension of beads in dIH₂O was applied to the surface of the microwell plate and the beads wereplaced into the microwells by centrifugation. Approximately 50% ofmicrowells contained 1 bead, approximately 20% of microwells contained 2or more beads and the remaining microwells were empty. The bead arraywas air-dried and subsequently exposed to 365 nm near-UV light for 15min through openings into the microwells in order to cleave thephotolabile linker between the peptide and the bead.

Example 35

Fabrication and Use of a Reference Microwell Plate for TestingConditions of Analyte Elution from a Bead Array Using EnzymaticDigestion

The bead array was fabricated as described in the previous EXAMPLE withthe exception that a peptide sequence conjugated to a TENTAGEL® bead was[FL]-GKGEAIYAAPFAKKKGGGSGGGG-PEG (SEQ ID NO: 27) and no UV photoreleasewas performed on the fabricated bead array. Instead, the peptide beadarray contained protease recognition sites for thermolysin and pronase(commercially available mixture of digestive enzymes).

Example 36

Fabrication of a Bead Array Featuring a Gap Between an Outer Layer of aBead and a Sidewall of a Microwell

The microwell array plate was fabricated from COP coated with aconductive surface layer of Indium Tin Oxide (ITO) and containedmicrowells approximately 250 μm wide and 250 μm deep. The TENTAGEL® MBNH₂ macrobead resins were from Rapp Polymere (Tubingen, Germany). Thebeads were placed into the microwells by centrifugation using thepreviously described techniques. FIG. 43A is a microphotograph of a beadarray, which was fabricated using particles with the size distributionbetween 140 and 170 μm, Rapp Polymere catalog number MB160002. FIG. 43Bis a microphotograph of a bead array, which was fabricated usingparticles with the size distribution between 200 and 250 μm, RappPolymere catalog number MB250002. Both images were acquired on a NIKONEclipse Ti instrument using brightfield microscopy. The images reveal abead array comprising a gap (i.e. spacing) between an outer layer of abead and a sidewall of a microwell. The unoccupied portion of amicrowell is accessible to an ionization beam of a MALDI massspectrometer and may be used to measure analyte(s), which have elutedfrom a bead into the microwell. In contrast, direct analysis of the beadsurface by MALDI MS may yield either weak or zero signal due to poorcompatibility of the polymer material of TENTAGEL® beads the with MALDIprocess. It is estimated that for MB160002 the portion of a microwell,which is not occupied by a bead, has dimensions greater than 50 μm,which is approximately equal to the diameter of a laser beam in aconventional MALDI TOF mass spectrometer. The bead array shown in FIG.43B has a larger portion of a microwell occupied by a bead and may besuitable for the mass spectrometric methods of analysis, which canperform analyte desorption directly from the surface of a bead,preferably at ambient pressure. Examples of such techniques are DESI andLMJ-SSP.

Example 37

Optical Image-Directed Acquisition of the Mass Spectrometric Data from aBead Array

An optical image of the bead array fabricated on a microwell array plateas described in the previous Example is shown in FIG. 44A. The opticalimage was processed using a custom-developed software algorithm toidentify areas comprising a gap between a bead in a microwell and asidewall of the microwell. The identified areas are schematicallydepicted in FIG. 44B as white squares measuring approximately 80×80micron, which are superimposed on the bead array image.

The identified array of squares shown in FIG. 44C was subsequently usedto guide acquisition of the MALDI TOF MS data from the microwell plate.The MS data acquisition was limited to regions coinciding with thelocations and dimensions of the areas identified in the previous stepand within each area a total of 9 mass spectra were acquired in thestationary mode and subsequently averaged. The mass spectra wereindependently acquired from locations separated by 40 μm in both X and Ydirections within the 80×80 μm area. In contrast to the MS imagingapproach, which can scan the entire surface of the microwell plate, thedescribed technique can provide significant time savings because onlythe bead-occupied microwells are measured and within each microwell themass spec data is acquired in locations most likely to yield a strongsignal.

Example 38

Fabrication of a Bead Array from a Library of Fluorescent Magnetic Beads

The microwell array plate was the same type as described in the previousExample. The fluorescent yellow carboxyl magnetic beads were fromSpherotech Inc (Lake Forest, Ill.), the bead catalog numberFCM-200052-2. The nominal size of magnetic beads was between 180 and 210μm. A bead suspension containing approximately 1000 microbeads in dI H₂Owas applied to the surface of the microwell array plate. An ALNICOmagnet bar measuring 50×6×6 mm (Fisher Scientific; catalog number543020) was placed underneath the microwell array plate and moved at thespeed of about 1 mm/sec in a spiral-like motion. The beads generallyfollowed the magnet movement direction on the surface of the microwellplate before encountering and sinking into empty microwells. Using thedescribed technique, a bead array comprising approximately 1000microbeads localized within a 10×10 mm area at 1 bead per well occupancywas fabricated in less than 2 minutes.

Example 38

Photolabile Peptide Bead Mass Tags Identifiable by Multiple Peaks inMass Spectra

A peptide mass tag RPPGFSRFRGGGSGGSG (SEQ ID NO: 24) conjugated to 90 μmdiameter TENTAGEL® beads via a 365 nm Fmoc photolabile linker wassynthesized on the beads using standard protocols of Fmoc chemistry. Thesynthesized peptide was used without further purification. MALDI TOFmass spectra were acquired from individual or multiple beads after themass tag photorelease from the beads positioned on a MALDI-compatiblesurface followed by addition of 10 mg/mL CHCA matrix solution. Theacquired mass spectra are shown in FIG. 45. The mass spectra containedthe isotopic envelope with a monoisotopic peak at 1634.7 m/z due to thefull-length peptide and in addition several lower intensity peaks near1520.7, 1491.7, 1290.6 and 1176.5 m/z, all of which exhibited thesignal-to-noise (SNR) ratio of greater than 250:1. The abovementionedpeaks are labeled with an arrow in FIG. 45. In this Example, thespecific peptide mass tag (and therefore the corresponding bead to whichthe tag was originally bound) can be identified not only by a singlepeak (in this case 1634.7 m/z), but by a combination of two or morepeaks comprising the mass tag “spectral signature”. The described methodenables greater confidence in the identification of a particular masstag within a bead array because several peaks can be detected thereforeminimizing the possibility of a spectral overlap. In addition, compoundswith the purity of less than 90% and sometimes with the purity of lessthan 50% may be utilized using the described approach.

Example 39

Dual Fluorescence and MS Readout from Bead Arrays Fabricated on SU-8Photoresist Coated Microwell Fiber Optic Plates

The SU-8 coated microwell array plates were from INCOM (Charlton,Mass.), as described in MATERIALS AND METHODS. To ensure compatibilitywith MALDI TOF mass spectrometry, the microwell plates were coated withan approximately 10 nm thick layer of Indium Tin Oxide (ITO) as aservice provided by Thin Films Inc (Hillsboro, N.J.). FIGS. 46A and 46Bdepict the structure of individual microwells as determined by confocalmicroscopy. FIG. 46C is an image of a bead inside a microwell acquiredon a fluorescence microscope in the “face up” plate orientation. In thisexperiment the fluorescent analyte was localized on the bead. It isnoted that in addition to the mass spectrometric readout, the fiberoptic faceplate enables imaging of the contents of a single microwell(e.g. a microparticle or a biological cell) through multiple opticfibers, which in this example are 6 μm wide but can be 3 μm or even 1 μmwide. It is therefore possible to obtain an image of a microparticlewith spatial resolution as high as 1 micron. Such capability may beuseful when a particle is image-encoded, e.g. contains an opticalbarcode or a combination of fluorescent dyes localized at differentlayers within the particle core.

Example 40

Optically Encoded Beads for Multiplexed Mass Spectrometric Bioassays

Bead kits comprising several populations of optically distinctsurface-activated microbeads suitable for: (i) conjugation ofbiomolecules, (ii) performing multiplexed reactions involving thebead-conjugated compounds in a bead suspension and/or bead array formatsand (iii) downstream analysis of the biomolecular reactions involvingthe bead-conjugated compounds using mass spectrometry were prepared fromcommercially available and custom fabricated bead stocks. Thefluorescent polystyrene beads were from Phosphorex, Inc (Hopkinton,Mass.). The beads were dyed with either a single fluorescent dye or acombination of two fluorescent dyes using the conventional dyeentrapment technique, i.e. the polymer beads were swollen in an organicsolvent to allow entry of the hydrophobic fluorescent dye(s) into thebead core and subsequently transferred into an aqueous medium, whichcaused the polystyrene matrix to collapse thus effectively trapping thefluorescent dye(s) within the bead core. Within each bead population,the beads were essentially monodisperse, i.e. the CV was less than 10%.The bead surface was either hydrophobic polystyrene suitable forantibody, protein and peptide attachment through non-specificadsorption, or carboxyl (COOH) suitable for ligand coupling throughprimary amine groups. Specific characteristics of the fabricated beadkits are shown in Table 2. Optical properties of the fluorescent dyesused in fabrication of Kit 1 and Kit 2 are shown in FIG. 47A and FIG.47B, respectively. The beads in Kit 1 have different fluorescenceabsorption and emission spectra. The beads in Kit 2 have differentcombinations of two fluorescent dyes, i.e. green only, orange only andgreen-orange. The beads in Kit 3 are encoded both by the bead size andby the bead fluorescence properties. The microwell array plates used forscreening of the fabricated bead kits comprise 120 μm wide, 120 μm deepmicrowells, which can accept both the 75 μm and 100 μm diameter beads atone bead per well ratio. Imaging of the fabricated bead array on aninverted fluorescence microscope, such as NIKON Eclipse Ti allowedidentification of the individual beads based on their optical propertiesand the dimensions.

TABLE 2 Dimensions, surface properties and optical properties ofoptically encoded bead libraries suitable for development of multiplexedmass spectrometric on-bead and off-bead assays Bead Color Bead Size BeadSurface KIT 1 Blue 100 μm polystyrene Green 100 μm polystyrene Orange100 μm polystyrene KIT 2 Green  75 μm polystyrene Orange  75 μmpolystyrene Orange-Green  75 μm polystyrene KIT 3 Blue 100 μm COOH Green100 μm COOH Orange 100 μm COOH Green  75 μm COOH Orange  75 μm COOHOrange-Green  75 μm COOH

Example 41

Optically Encoded Beads for Multiplexed Mass Spectrometric Bioassays

A bead kit comprising several populations of optically distinctmicrobeads encoded by a combination of two fluorescent dyes mixed in apre-determined ratio was fabricated and subsequently measured by massspectrometry and fluorescence imaging.

The beads were plain polystyrene beads in the 106-125 μm diameter rangeavailable from Bangs Laboratories, Inc (Fishers, Ind.). The Dragon Greenand Flash Red fluorescent dyes and their optical properties areavailable from Bangs Labs. The beads were labeled with a combination ofthe two dyes using the standard solvent swelling and dye entrapmentprocess. The dye ratios used for the bead fabrication are provided inTable 3. The listed ratios refer to the dye input, not the ratio offluorescence intensities obtained from the individual dyed beads,although achieving sufficiently precise ratios of fluorescenceintensities is also possible.

TABLE 3 Beads encoded by a combination of two fluorescent dyes. BeadType Dragon Green Flash Red 1 0 100%  2 25% 75% 3 33% 67% 4 50% 50% 567% 33% 6 75% 25% 7 100%  0

Bead arrays featuring the bead types selected from Table 3 werefabricated on fiber optic, fused silica and APEX™ glass microwell platesincluding the ITO-coated plates and analyzed on the NIKON Eclipse Tiinverted microscope system. The optical data was acquired from the beadarrays in the “face up” and “face down” microwell plate configurationusing FITC, Texas Red and Cy5 filter channels. In some cases, 3D(three-dimensional) fluorescence imaging of the bead array was performedusing the depth profiling option of the NIKON NIS Elements version 4.13acquisition software. The Z drive step was 0.9 μm when performing thedepth profiling. When performing the 3D imaging of the bead array, theintensity used in subsequent calculations was measured at certain Zslices, alternatively the intensity for the entire bead wasvolume-integrated within the NIS Elements software. The intensity ofacquired fluorescence signal in each channel was statistically analyzedfor bead populations comprising up to 1,000 beads to obtain the mean andstandard deviation values. It was concluded that each bead type listedin Table 3 can be reliably, i.e. unambiguously identified on the basisof the ratio of fluorescence intensities recorded from individual beadsin a bead array format. The fluorescence analysis of bead arraysfeaturing optically encoded beads can be performed using a fluorescencemicroscope or a fluorescence microarray scanner.

Example 42

Three-Dimensional Fluorescence Imaging of a Reacted Bead Array Followedby Mass Spectrometry Analysis

Fluorescent peptide [5-FAM]VFDZGGGSGGSG-PLL-Ahx-TG Bead (SEQ ID NO: 23),where [5-FAM] is 5-carboxyfluorescein, PLL is a photolabile linkercleavable by 365 nm light, Ahx is an aminohexanoic acid spacer, Z is amixture of 19 amino acids and TG Bead is a TENTAGEL® 90 μm diameter beadwas used to fabricate a bead array on a microwell array plate. Thepeptide bead array was exposed to a dilute (1 mg/mL) solution of trypsindelivered to the surface of the microwell plate as an aerosol. In theabsence of UV exposure, the peptide digestion with trypsin is expectedto release only a fraction of the fluorescent label from the bead, e.g.the peptides containing either Arg or Lys in the Z position.

The reacted bead array was subsequently imaged on NIKON Eclipse Tifluorescence microscope using the 3D imaging (depth profiling) option.Several representative images spaced apart by ˜3 μm are shown in FIGS.48A-48C. The acquired fluorescence data was subsequently used todetermine localization of the eluted peptide analyte on the top surfaceof the microwell plate and to restrict acquisition of the mass spectraldata to areas containing the eluted analyte.

Example 43

Dual Fluorescence and Mass Spectrometric Readout from Individual Cellswithin a Cell Microarray on a Microwell Plate

The MCF-7 cell line cells were transfected with eGFP (enhanced greenfluorescent protein) using the LIPOFECTAMINE® transfection protocol. Inorder to measure efficiency of the transfection reaction, a cellsuspension containing greater than 10,000 transfected cells in PBS wasapplied to the surface of a microwell array plate and the cells weredistributed into individual microwells by centrifugation. The microwellplate was Rectangular Fiberoptic Faceplate from INCOM (Charlton, Mass.),75.0 mm×25.0 mm×1.0 mm Thick; material is Block Press BXI84-50, withinterstitial EMA, 50 micron fiber size, one side etched to 30 microndepth. The microwell plate contains over 750,000 individual microwellswithin dimensions of the standard microscope slide, each microwellfunctionally connected to a single optic fiber. The area used forfabrication of the cell array contained approximately 100,000microwells. The fabricated cell array on the microwell plate was imagedon a fluorescence microscope (NIKON Eclipse Ti) via optic fibers. Asshown in FIG. 49A, microwells containing non GFP-expressing cells andempty microwells do not generate detectable fluorescence signal. Incontrast, as shown in FIG. 49B, microwells containing a singleGFP-expressing cell can be visualized by their above-backgroundfluorescence signal.

Following acquisition of the fluorescence image data, the cell array wascoated with CHCA MALDI matrix solution using the aerosol method ofmatrix deposition and analyzed by MALDI TOF mass spectrometry. Briefly,the hexagonal array of microwells within the microwell array plate wasused to generate a grid of sample spots to direct the MS instrument (ABSciex 4800 MALDI TOF-TOF Analyzer) to acquire MS data from the center ofeach microwell, regardless of whether the microwell contained a cell.Each mass spectrum was collected in the linear positive mode, in themass range between 200 and 800 m/z, from a stationary position andcontained an average of 50 single-shot spectra. Several peaks previouslydetected in whole cell mass spectra of single cells were detected inlocations coinciding with locations exhibiting above the backgroundfluorescence signal (GFP transfected cells), as well as some locationsexhibiting the background fluorescence signal (non-transfected cells).The detected signals were likely due to certain cell metabolites andcommon lipids. Peaks characteristic of the CHCA matrix were detectedthroughout the microwell plate but did not significantly overlap withthe observed analyte peaks. As shown in Table 4, the greater number ofcells detected by mass spectrometry indicates that efficiency of thetransfection reaction was approximately 40%.

TABLE 4 The total approximate number of cells in a cell array and thenumber of cells detected by fluorescence and WCMS. Expected NumberDetected by Detected by of Cells Fluorescence Mass Spec ~10,000 ~3,800~8,600

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While thepresent disclosure has been described in connection with the specificembodiments thereof, it will be understood that it is capable of furthermodification. Furthermore, this application is intended to cover anyvariations, uses, or adaptations of the disclosure, including suchdepartures from the present disclosure as come within known or customarypractice in the art to which the disclosure pertains, and as fall withinthe scope of the appended claims.

REFERENCES

-   Boggio, K. J., E. Obasuyi, K. Sugino, S. B. Nelson, N. Y. Agar,    and J. N. Agar. 2011. “Recent advances in single-cell MALDI mass    spectrometry imaging and potential clinical impact.” Expert Rev    Proteomics 8:591-604.-   Braeckmans, K., S. C. De Smedt, M. Leblans, R. Pauwels, and J.    Demeester. 2002. “Encoding microcarriers: present and future    technologies.” Nat Rev Drug Discov 1:447-56.-   Dong, H., W. Shen, M. T. Cheung, Y. Liang, H. Y. Cheung, G.    Allmaier, O. Kin-Chung Au, and Y. W. Lam. 2011. “Rapid detection of    apoptosis in mammalian cells by using intact cell MALDI mass    spectrometry.” Analyst 136:5181-9.-   Gagnaire, J., O. Dauwalder, S. Boisset, D. Khau, A. M. Freydiere, F.    Ader, M. Bes, G. Lina, A. Tristan, M. E. Reverdy, A. Marchand, T.    Geissmann, Y. Benito, G. Durand, J. P. Charrier, J. Etienne, M.    Welker, A. Van Belkum, and F. Vandenesch. 2012. “Detection of    Staphylococcus aureus Delta-Toxin Production by Whole-Cell MALDI-TOF    Mass Spectrometry.” PLoS One 7:e40660.-   Hanrieder, J., G. Wicher, J. Bergquist, M. Andersson, and A.    Fex-Svenningsen. 2011. “MALDI mass spectrometry based molecular    phenotyping of CNS glial cells for prediction in mammalian brain    tissue.” Anal Bioanal Chem 401:135-47.-   Hazen, T. H., R. J. Martinez, Y. Chen, P. C. Lafon, N. M.    Garrett, M. B. Parsons, C. A. Bopp, M. C. Sullards, and P. A.    Sobecky. 2009. “Rapid identification of Vibrio parahaemolyticus by    whole-cell matrix-assisted laser desorption ionization-time of    flight mass spectrometry.” Appl Environ Microbiol 75:6745-56.-   Kulkarni, M. J., V. P. Vinod, P. K. Umasankar, M. S. Patole, and M.    Rao. 2006. “Intact cell matrix-assisted laser desorption/ionization    mass spectrometry as a tool to screen drugs in vivo for regulation    of protein expression.” Rapid Commun Mass Spectrom 20:2769-72.-   Pantano, P., and D. R. Walt. 1996. “Ordered nanowell arrays.”    Chemistry of Materials 8:2832-2835.-   Urban, P. L., K. Jefimovs, A. Amantonico, S. R. Fagerer, T.    Schmid, S. Madler, J. Puigmarti-Luis, N. Goedecke, and R.    Zenobi. 2010. “High-density micro-arrays for mass spectrometry.” Lab    Chip 10:3206-9.-   Wilson, R., A. R. Cossins, and D. G. Spiller. 2006. “Encoded    microcarriers for high-throughput multiplexed detection.” Angew Chem    Int Ed Engl 45:6104-17.-   Zhou, G., F. Khan, Q. Dai, J. E. Sylvester, and S. J. Kron. 2012.    “Photocleavable peptide-oligonucleotide conjugates for protein    kinase assays by MALDI-TOF MS.” Mol Biosyst 8:2395-404.-   Ziauddin, J., and D. M. Sabatini. 2001. “Microarrays of cells    expressing defined cDNAs.” Nature 411:107-10.

The instant application contains a Sequence Listing which is beingsubmitted electronically in ASCII format on even date herewith and ishereby incorporated by reference in its entirety. Said ASCII copy,created on Apr. 13, 2017, is named 83421C_SL.txt and is 8,836 bytes insize.

What is claimed is:
 1. A method of performing elution of an analyte froma bead array, the method comprising the steps of: providing a beadarray, the bead array comprising a solid support, a bead and an analyteassociated with the bead, wherein the solid support comprises amicrowell and the bead is submerged in the microwell; and causingmigration of the analyte from the bead onto at least one of a bottomsurface of the microwell, a sidewall of the microwell, and a surfaceoutside the microwell that is adjacent to the microwell, wherein thecausing migration step comprises exposing the solid support to anaerosol.
 2. The method of claim 1 wherein a diameter of the microwell isless than 1 mm.
 3. The method of claim 1 wherein a diameter of themicrowell exceeds a diameter of the bead by at least 10 microns.
 4. Themethod of claim 1 further comprising a step of delivering a layer ofMALDI matrix to the bottom surface of the microwell.
 5. The method ofclaim 1 wherein the analyte is selected from a group consisting ofpeptides, peptoids, peptidomimetics, proteins, antibodies, metabolites,lipids, carbohydrates, nucleic acids and drug compounds.
 6. The methodof claim 1 wherein the analyte comprises two chemically distinctpeptides.
 7. The method of claim 1 further comprising the step ofreducing dimensions of the bead while the bead remains disposed in themicrowell.
 8. The method of claim 1 further comprising the step ofremoving the bead from the solid support.
 9. The method of claim 8wherein the bead removing step comprises exposing the bead to a streamof gas.
 10. The method of claim 1 wherein the sidewall of the microwellis hydrophobic.
 11. The method of claim 1 wherein the sidewall of themicrowell comprises a polymer.
 12. The method of claim 1 wherein thesidewall of the microwell comprises silicone.
 13. The method of claim 1further comprising the step of depositing a layer of MALDI matrix ontothe solid support.
 14. A method of performing elution of an analyte froma bead array, the method comprising the steps of: providing a beadarray, the bead array comprising a solid support, a bead and an analyteassociated with the bead, wherein the solid support comprises amicrowell and the bead is submerged in the microwell, wherein theanalyte comprises two chemically distinct peptides, and wherein the twochemically distinct peptides are selected from the group consisting ofpeptides with different isotope abundance, peptides with differentfluorescent labels, peptides with at least one amino acid substitutionin an otherwise identical amino acid sequence and peptides with adifferent number or different positions of post-translationalmodifications (PTMs) in an otherwise identical amino acid sequence; andcausing migration of the analyte from the bead onto at least one of abottom surface of the microwell, a sidewall of the microwell, and asurface outside the microwell that is adjacent to the microwell.
 15. Amethod of performing elution of an analyte from a bead array, the methodcomprising the steps of: providing a bead array, the bead arraycomprising a solid support, a bead and an analyte associated with thebead, wherein the solid support comprises a microwell and the bead issubmerged in the microwell, causing migration of the analyte from thebead onto at least one of a bottom surface of the microwell, a sidewallof the microwell, and a surface outside the microwell that is adjacentto the microwell, and repositioning the analyte on the solid support,the repositioning step being performed after the causing migration stepand comprising dissolving and subsequently precipitating the analyte onthe solid support whereby localization of the analyte on the solidsupport changes.